1. Developmental Biology
  2. Stem Cells and Regenerative Medicine
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Stem cell topography splits growth and homeostatic functions in the fish gill

  1. Julian Stolper
  2. Elizabeth Mayela Ambrosio
  3. Diana-Patricia Danciu
  4. Lorena Buono
  5. David A Elliott
  6. Kiyoshi Naruse
  7. Juan R Martínez-Morales
  8. Anna Marciniak-Czochra
  9. Lazaro Centanin  Is a corresponding author
  1. Heidelberg University, Germany
  2. Royal Children’s Hospital, Australia
  3. Universidad Pablo de Olavide, Spain
  4. National Institute for Basic Biology, National Institutes of Natural Sciences, Japan
Research Article
Cite this article as: eLife 2019;8:e43747 doi: 10.7554/eLife.43747
8 figures, 4 videos, 1 table, 1 data set and 7 additional files

Figures

Growth and homeostasis in the medaka gill.

(A) Enucleated entire gills of medaka at different post-embryonic times show that organ size increases during post-embryonic growth (left). A gill contains four pairs of branchial arches (middle left) that display numerous filaments (middle right). Filaments are composed of lamella (right), where gas exchange occurs. (B) Scheme depicting that branchial arches grow by increasing the number of filaments, and filaments grow by increasing its length. (C) The number of filaments per branchial arch is higher in bigger fish - x axis represents fish length, and y axis the number of filaments in the second right branchial arch. (D) IdU incorporation in the adult gill reflects proliferating cells all along the longitudinal axis of a filament. Scale bars are 100 µm in (A) filament, and 20 µm in (A) lamella and (D).

https://doi.org/10.7554/eLife.43747.002
Gill stem cells located at the periphery of branchial arches generate more filaments life-long.

(A) Scheme showing the expected outcome assuming a progenitor (left bottom) or a stem cell (right bottom) model. Please note that the schemes considered as ‘labelled’ any filament containing EGFP+ cells all along their longitudinal axis. (B) Entire gill from a double transgenic GaudíUbiq.iCre GaudíRSG fish 2 month after induction with TMX. (C) Branchial arch from a double transgenic GaudíUbiq.iCre GaudíRSG fish 2 months after induction with TMX. Arrowheads in B and C indicate recombined embryonic filaments located at the centre of branchial arches, and asterisks indicate stretches of peripheral filaments with the same recombination status. Note that the present resolution does not allow revealing different recombination patterns in each filament. (D) Graphs showing the distribution of switches in stretches of the six most peripheral filaments. The graphs show a comparison of the experimental data (black) to the expected distribution according to a progenitor model (light gray, left) and to a stem cell model (gray, right). Scale bar is 500 µm in (C).

https://doi.org/10.7554/eLife.43747.003
Figure 3 with 1 supplement
Filament growth stem cells are located at the apical tip.

(A) Scheme showing the expected outcome of IdU pulse and chase experiments depending on the location of growth stem cells. (B) IdU pulse and chase experiment shows the apical region devoted of signal, indicating these cells were generated after the IdU pulse. (C) Scheme showing the expected outcome of a filament in which growth stem cells were labelled. (D) A filament from a double transgenic GaudíUbiq.iCre GaudíRSG fish one month after induction with TMX shows an expanding clone in the apical region, indicating a high proliferative activity compared to clones located at other coordinates along the longitudinal axis. (E, F). Scheme (E) and data (F) showing an IdU pulse and chase experiment on branchial arches. The apical part of each filament and the more peripheral filaments are devoted of signal revealing the stereotypic growth of branchial arches. (G) Scheme showing the manual sorting of apical and middle regions for total RNA preparation. RNA-seq revealed differentiated cells in the middle region of filaments and undifferentiated cells at the apical domain. Scale Bars are 20 μm in (B) and (D), and 100 μm in (F).

https://doi.org/10.7554/eLife.43747.004
Figure 3—source data 1

Transcriptome of apical and medial domains in a gill filament.

https://doi.org/10.7554/eLife.43747.006
Figure 3—figure supplement 1
Technical analysis of the RNA-seq data.

(A) Principal Component Analysis (PCA) plot of the three biological replicates of transcripts from the apical and the medial part of the medaka gill filaments. (B) Volcano-plot representation of differential expression analysis of the genes identified from the RNA-seq libraries of the apical and the medial part of the gill filaments. Red points mark the genes with change in expression between the two conditions with a p-value<0.05.

https://doi.org/10.7554/eLife.43747.005
Filament growth stem cells are fate restricted.

(A–B) A gill (A) and a branchial arch (B) from a double transgenic GaudíUbiq.iCre GaudíRSG fish two month after induction with TMX. (C–F) Confocal images from filaments in A, B, stained for EGFP and DAPI to reveal the cellular composition of different clones. Four different recombination patterns were identified. (G, G’) A detailed view of pattern 1(C) shows recombined epithelial cells covering each lamella. (H) Co-staining with an anti-Na+K+ATP-ase antibody confirms that MRC cells are clonal to other epithelial cells in the filament. (I, I’) Cross-section of a filament that displays pattern 2 (D). DAPI staining allows identifying blood cells (strong signal, small round nuclei), pillar cells (weaker signal, star-shaped nuclei), and chondrocytes (elongated nuclei at the central core of the filament) (I). The lineage tracker EGFP reveals that chondrocytes and pillar cells are clonal along a filament (I’). Scale Bars are 500 µm in (B), 20 µm in (C–H) and (I).

https://doi.org/10.7554/eLife.43747.007
Figure 5 with 1 supplement
Branchial arch stem cells are fate restricted.

(A) Scheme showing the expected outcome assuming that br-archSCs are fate restricted (middle) or multi-potent (bottom). The recombination pattern of consecutive filaments would be identical if generated by fate restricted br-archSCs, and non-identical if derived from a multipotent br-archSC. (B–E) Confocal images show an identical recombination pattern in consecutive peripheral filaments for pattern 1 (B), pattern 2 (C), pattern 3 (D) and pattern 4 (E). Scale Bars are 200 μm in (B).

https://doi.org/10.7554/eLife.43747.011
Figure 5—figure supplement 1
Early embryonic recombination and transplantations at blastula stage indicate fate-restricted stem cells in the fish gill.

(A) Consecutive filaments from a double transgenic GaudíHsp70A.Cre GaudíRSG fish 3 months after induction (heat-shock at stage 20). Filaments display recombination in specific patterns (pattern 2 in two filaments at the left, pattern 3 in three filaments at the right). (B) A Branchial arch from a double transgenic GaudíHsp70A.Cre GaudíRSG fish 3 months after induction (heat-shock at stage 20). All filaments are labelled in pattern 4 indicating that the earliest founder cell of that lineage was labelled. (C) Consecutive filaments of a chimeric fish generated by transplantations at blastula stage (Host: unlabelled WT, Donor: GaudiBBW - driving ubiquitously a membrane-tagged Cerulean fluorescent protein). Filaments display the recombination Pattern 2 - compare to Figure 4D and mind differences in nuclear vs membrane label. Scale Bars are 200 μm in (A, B), and 100 μm in (C).

https://doi.org/10.7554/eLife.43747.012
Figure 6 with 1 supplement
p53 Coordinates growth stem cells in a lineage-specific manner.

(A) Branchial arch of a p53-/- -to-WT chimera, where p53-/- mutant cells are labelled in green. Composite filaments display the proper length as compared to non-labelled neighbour filaments. (B) Branchial arch of a WT-to-p53-/- chimera, where WT cells are labelled in green. Composite filaments are shorter than their neighbours only when pattern 2 comes from the donor - Note the short size of the right filament in B’-B’’ compared to the filament at the left. Scale Bars are 500 μm in (A, B) and 100 μm in (B’–B’’’).

https://doi.org/10.7554/eLife.43747.013
Figure 6—figure supplement 1
Branchial Arch of p53E241X mutant.

(A) DAPI staining on a branchial arch extracted from a p53 homozygous mutant showing the un-distinguishable organisation, length and size of filaments and lamellae than wild-type samples.

https://doi.org/10.7554/eLife.43747.014
Homeostatic stem cells locate to the base of each lamella.

(A) DAPI image of peripheral filaments indicating the increasing number of lamellae per filament. (B) DAPI image of consecutive lamellae along a filament reveals that lamellae do not increase their size. (C) IdU pulse reveals proliferative cells at the base of the lamellae. (D–E) EGFP cells indicating clonal progression of clones in double transgenic GaudíUbiq.iCre GaudíRSG fish 1 month after induction with TMX during adulthood. Clones of pillar cells progress from the base to the distal part of a lamellae (D’’, E). Scale Bars are 200 μm in (A), and 20 μm in (B–E).

https://doi.org/10.7554/eLife.43747.015
The homeostatic domain sustains growth after filament ablation.

(A) Scheme of the ablation procedure. The growth domain and the upper part of the homeostatic domain are mechanically ablated. (B) DAPI image of control filaments shows an intact growth domain at the top. (C) DAPI image of injured filaments after a chase of 1 month shows a regenerated growth domain. (D) During the duration of the experiment, ablated filaments were unable to reach the length of their neighbour, non-ablated filaments. (E) IdU-positive cells are detected at the growth domain of both intact and ablated treatment. (F–I) Transmitted (F) and fluorescent (G–I) images of gill filaments from recombined GaudíUbiq.iCre GaudíRSG fish, 3 weeks post-ablation. The upper fraction containing the growth domain was generated after the ablation (highlighted in F). The recombination pattern observed in the regenerated zone is identical to the recombination pattern that the filaments had before ablation, indicating that cells maintain their fate during the regenerative response. Scale Bars are 500 µm in (A) and 100 µm in (C–I).

https://doi.org/10.7554/eLife.43747.017

Videos

Video 1
3D reconstruction of a pattern 3-labelled filament.

A middle section of filament in an adult GaudíUbiq.iCre GaudíRSG fish that was induced for recombination at late embryonic stages. The filament shows the lineage of a growth stem cell that labels pattern 3.

https://doi.org/10.7554/eLife.43747.008
Video 2
3D reconstruction of a pattern 4-labelled filament.

A middle section of filament in an adult GaudíUbiq.iCre GaudíRSG fish that was induced for recombination at late embryonic stages. The filament shows the lineage of a growth stem cell that labels pattern 4.

https://doi.org/10.7554/eLife.43747.009
Video 3
3D reconstruction of a pattern 2-labelled filament.

A middle section of filament in an adult GaudíUbiq.iCre GaudíRSG fish that was induced for recombination at late embryonic stages. The filament shows the lineage of a growth stem cell that labels pattern 2.

https://doi.org/10.7554/eLife.43747.010
Video 4
3D reconstruction of a pattern 2-labelled lamella.

A middle section of filament in an adult GaudíUbiq.iCre GaudíRSG fish that was induced for recombination at late embryonic stages. The filament shows the lineage of a homeostatic stem cell that labels pattern 2 only in one lamella.

https://doi.org/10.7554/eLife.43747.016

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain
background
(Oryzias latipes)
Cabwild type stock,
southern population
Genetic
reagent (O. latipes)
p53 E241X mutantTaniguchi et al., 2006
Genetic
reagent (O. latipes)
GaudíUbiq.iCreCentanin et al., 2014
Genetic
reagent (O. latipes)
GaudíHsp70.ACentanin et al., 2014
Genetic
reagent (O. latipes)
GaudíRSGCentanin et al., 2014
Genetic
reagent (O. latipes)
GaudiLoxPOUTCentanin et al., 2014
Genetic
reagent (O. latipes)
GaudiBBWCentanin et al., 2014
Antibodya-EGFP
(Rabbit IgG polyclonal)
Invitrogen
(now Thermo Fischer)
CAB4211;
RRID: AB_10709851
Dilution 1:750
Antibodya-EGFP
(Chicken IgY polyclonal)
life technologiesA10262;
RRID: AB_2534023
Dilution 1:750
Antibodya-Na + K + ATP-ase
(Rabbit monoclonal)
Abcamab76020,
EP1845Y
Dilution 1:200
Antibodya-BrdU/IdU
(Mouse IgG monoclonal)
Becton Dickinson347580Dilution 1:50
AntibodyAlexa 488 Goat
a-Rabbit
Invitrogen
(now Thermo Fischer)
A-11034Dilution 1:500
AntibodyAlexa 488 Donkey
a-Chicken
Invitrogen
(now Thermo Fischer)
703-545-155Dilution 1:500
AntibodyAlexa 647 Goat
a-Rabbit
Life TechnologiesA-21245Dilution 1:500
AntibodyAlexa 647 Goat
a-Rabbit
Life TechnologiesA-21245Dilution 1:500
AntibodyCy5 Donkey
a-Mouse
Jackson715-175-151Dilution 1:500
Chemical
compound, drug
tamoxifenSigmaT5648
Chemical
compound, drug
tricaineSigma-AldrichA5040-25G
Chemical
compound, drug
BrdUSigma-AldrichB5002final concentration
of 0,4 g/l
Chemical
compound, drug
IdUSigma-AldrichI7125final concentration
of 0,4 g/l
Chemical
compound, drug
Trizol
Software,
algorithm
EnsemblePublic
OtherDAPIRothfinal concentration
of 5 ug/l
Software,
algorithm
DAVID 6.8https://david.ncifcrf.gov/home.jsp

Data availability

All data analysed for this study is included in the manuscript and supporting files. Raw sequencing data have been deposited in GEO under accession code GSE130939.

The following data sets were generated
  1. 1
    NCBI Gene Expression Omnibus
    1. L Buono
    2. JR Martinez-Morales
    3. L Centanin
    (2019)
    ID GSE130939. Transcriptome analysts of a growth domain and a differentiated domain of the medaka gill.

Additional files

Supplementary file 1

Simulation of recombined pattern in branchial arches assuming progenitor cells.

The table represents simulated values of recombination (0 = unlabelled; 1 = labelled) in five gills, according to the ‘progenitor’ model described in M and M. The labelling efficiency was calculated from real data from 5 GaudíUbiq.iCre GaudíRSG recombined gills.

https://doi.org/10.7554/eLife.43747.018
Supplementary file 2

Simulation of recombined pattern in branchial arches assuming stem cells.

The table represents simulated values of recombination (0 = unlabelled; 1 = labelled) in five gills, according to the ‘stem cell’ model described in M and M. The labelling efficiency was calculated from real data from 5 GaudíUbiq.iCre GaudíRSG recombined gills.

https://doi.org/10.7554/eLife.43747.019
Supplementary file 3

Experimental data of recombined pattern in adult branchial arches.

The table includes values for recombination (0 = unlabelled; 1 = labelled) in five gills enucleated from adult GaudíUbiq.iCre GaudíRSG fish that were induced for recombination at late embryonic stages.

https://doi.org/10.7554/eLife.43747.020
Supplementary file 4

Objective function representation comparing data to progenitor and stem cell models.

The table displays the values for an objective function, comparing the recombination pattern obtained in 22 GaudíUbiq.iCre GaudíRSG recombined gills with the predicted values for a stem cell or a progenitor model (See M and M). Lower values for the objective function represent a better fit between experimental and simulated data.

https://doi.org/10.7554/eLife.43747.021
Supplementary file 5

Enriched transcripts in the apical and medial domains of gill filaments.

Representation of transcripts obtained from the apical and the medial domain of gill filaments, ordered according to their differential expression.

https://doi.org/10.7554/eLife.43747.022
Supplementary file 6

Experimental data of recombined patterns in adult branchial arches with cellular resolution.

The table displays values of recombination (0 = unlabelled; 1 = Pattern 1; 2 = Pattern 2; 3 = Pattern 3; 4 = Pattern 4) in gills enucleated from adult GaudíUbiq.iCre GaudíRSG fish that were induced for recombination at late embryonic stages.

https://doi.org/10.7554/eLife.43747.023
Transparent reporting form
https://doi.org/10.7554/eLife.43747.024

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