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Ttc7a regulates haematopoietic stem cell functions while controlling the

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DOI: 10.3324/haematol.2018.207100

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 Published Ahead of Print on April 19, 2019, as doi:10.3324/haematol.2018.207100.
Copyright 2019 Ferrata Storti Foundation.

Ttc7a regulates haematopoietic stem cell functions while

controlling the stress-induced response

by Claire Leveau, Tania Gajardo, Marie-Thérèse El-Daher, Nicolas Cagnard, Alain Fischer,
Geneviève de Saint Basile, and Fernando E. Sepulveda

Haematologica 2019 [Epub ahead of print]

Citation: Claire Leveau, Tania Gajardo, Marie-Thérèse El-Daher, Nicolas Cagnard, Alain Fischer,
Geneviève de Saint Basile, and Fernando E. Sepulveda. Ttc7a regulates haematopoietic stem cell
functions while controlling the stress-induced response.
Haematologica. 2019; 104:xxx
doi:10.3324/haematol.2018.207100

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Ttc7a regulates haematopoietic stem cell functions while controlling the stress-induced
response.

Claire Leveau1,2, Tania Gajardo1,2, Marie-Thérèse El-Daher1,2, Nicolas Cagnard2,3,4, Alain

Fischer2,5,6,7, Geneviève de Saint Basile1,2,8,* and Fernando E. Sepulveda1,2,9,*.

1
Laboratory of Normal and Pathological Homeostasis of the Immune System, INSERM
UMR 1163, Imagine Institute, Paris, France
2
Université Paris Descartes -Sorbonne Paris Cité, Imagine Institute, Paris, France
3
Bioinformatic Platform, INSERM UMR 1163, Université Paris Descartes-Sorbonne Paris
Cité, Imagine Institute, Paris, France
4
Structure Fédérative de Recherche (SFR) Necker, INSERM US24/CNRS UMS 3633, Paris,
France
5
Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades Immunology and
Pediatric Hematology Department, Paris, France
6
Collège de France, Paris, France
7
INSERM UMR1163, Paris, France
8
Assistance Publique-Hôpitaux de Paris, Hôpital Necker-Enfants Malades, Centre d'Etudes
des Déficits Immunitaires, Paris, France
9
Centre National de la Recherche Scientifique – CNRS
*
These authors contributed equally to this work

Short title: Ttc7a controls HSC functions.

Correspondence:
Geneviève de Saint Basile, MD, PhD Fernando E. Sepulveda, PhD
Inserm U1163, Imagine Institute Inserm U1163, Imagine Institute
24 Boulevard de Montparnasse 24 Boulevard de Montparnasse
F-75015 Paris, France F-75015 Paris, France
Phone: +33 142 754 335 Phone: + 33 142 754 410
Fax: +33 142 754 221 Fax: + 33 142 754 221
genevieve.de-saint-basile@inserm.fr fernando.sepulveda@inserm.fr
Leveau et al. Ttc7a controls HSC functions…

Abstract
The molecular machinery that regulates the balance between self-renewal and differentiation
properties of hematopoietic stem cells has yet to be characterized in detail. Here we found that
the tetratricopeptide repeat domain 7 A (Ttc7a) protein, a putative scaffold protein expressed
by hematopoietic stem cells, acts as an intrinsic regulator of the proliferative response and the
self- renewal potential of murine hematopoietic stem cells in vivo. Loss of Ttc7a consistently
enhanced the hematopoietic stem cells’ competitive repopulation ability and their intrinsic
capacity to replenish the hematopoietic system after serial cell transplantations, relative to
wild-type cells. Ttc7a-deficient hematopoietic stem cells exhibit a different transcriptomic
profile for a set of genes controlling the cellular response to stress, which was associated with
increased proliferation in response to chemically induced stress in vitro and myeloablative
stress in vivo. Our results therefore revealed a previously unrecognized role of Ttc7a as a
critical regulator of the hematopoietic stem cells stemness. This role is related, at least in part,
to regulation of the endoplasmic reticulum stress response.

Keywords
Ttc7a/HSC/ER stress/self-renewal.

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Leveau et al. Ttc7a controls HSC functions…

Introduction
In fsn mice, the spontaneous insertion of early transposon into the Ttc7a gene is known to
1, 2
impair Ttc7a protein expression . Consequently, fsn mice develop a proliferative lymphoid
and myeloid disorder, with hyperplasia of the spleen and lymph nodes, elevated monocyte,
granulocyte and lymphoid cell counts 3-6, and severe anaemia 7. Moreover, fsn mice exhibit a
reduced lifespan and changes in the skin (epidermal hyperplasia and inflammation) 8, 9 and the
intestinal tract (gastric papillomas) 10. The marked phenotype alterations in fsn mice suggest
that Ttc7a protein has one or more major regulatory roles in the hematopoietic system, and,
potentially, in other tissues of epithelial origin.
Ttc7a is a putative scaffolding protein as it contains nine tetratricopeptide repeats
(TPR) domains, that are predicted to interact with proteins containing their own TPRs or other
11
motifs . These TPR-containing proteins are involved in a variety of biological processes,
including cell cycle control, protein trafficking, secretion and protein quality control. Indeed,
TPR-containing proteins have been shown to bind chaperones such as Hsp90 and Hsp70,
controlling their activity 12-14. Thus, Ttc7a is likely to be involved in a broad range of protein
complexes and hence functions. In vitro studies have shown that the loss of Ttc7a causes
inappropriate activation of RhoA-dependent effectors and thus disrupts cytoskeletal dynamics
15, 16
. Furthermore, TTC7A reportedly interacts with EFR3 homolog B and the
phosphatidylinositol 4-kinase alpha, which is known to catalyse the production of
phosphatidylinositol 4-phosphate at the plasma membrane in yeast and human cells 17, 18. This
observation emphasizes the conservation, at least in part, of Ttc7a’s functions during
evolution. However, data on TTC7A’s biological function(s) are still scarce.
Inadequate proliferation of peripheral haematopoietic lineages has been reported in
several modified murine models; this impairment is ultimately associated with the exhaustion
of the haematopoietic stem cell (HSC) pool 19. Indeed, the production of blood cells requires
HSCs to leave their quiescent state and differentiate into functional progeny. An excessive
requirement for haematopoietic cell production biases HSC function toward differentiation, at
the expense of self-renewal 20. Various intrinsic and extrinsic factors influence the HSCs’ fate
decisions, i.e. quiescence or proliferation. Endoplasmic reticulum (ER) stress has recently
21
been highlighted as an important regulator of HSC function . This stress is triggered by
various stimuli and leads to the accumulation of unfolded proteins in the lumen of the ER, and
the induction of the unfolded protein response (UPR). The chaperone BIP (Hspa5/GRP78) is
the main inducer of the UPR 22. This response results in the enhanced expression of chaperone
proteins (heat shock proteins (Hsp)), phosphodiesterase (Pdi), and other proteins like

3
Leveau et al. Ttc7a controls HSC functions…

calreticulin that, together with BIP, boosts protein folding capacities. Depending on ER stress
intensity, UPR activation can lead to apoptosis or survival 23.
In the present study, we found that Ttc7a regulates the murine HSCs’ self-renewal and
haematopoietic reconstitution potential and controls their sensitivity to stress. Loss of Ttc7a
consistently enhanced the HSCs’ stemness, since Ttc7a-deficient HSCs displayed a greater
proliferation capacity than control counterparts in response to ER stress in vitro, and after
myeloablative stress in vivo. Hence, our present results unravel a new role for Ttc7a as a
regulator of self-renewal and response to stress in HSCs.

4
Leveau et al. Ttc7a controls HSC functions…

Methods
Mice. Heterozygous Balb/cByJ fsn (CByJ.A-Ttc7fsn/J) mice and Balb/cByJ CD45.1
(CByJ.SJL(B6)-Ptprca/J) mice were obtained from the Jackson Laboratory. All mice were
maintained in SPF (Specific Pathogen Free) and handled according to national and
institutional guidelines.

Repopulations assays. Bone marrow (BM) cells were transferred into CD45.1+ ctrl recipient
mice upon irradiation. 30,000 LSK donor cells were injected into irradiated recipient mice.
For serial transplantations, recipients were reconstituted with 107 BM cells. To perform
competitive repopulation assays, 1,000 LSK cells were injected with 2 x 106 unfractionated
CD45.1+ bone marrow cells. 12 weeks after transfer, mice were treated with a single dose of
5-FU (150 mg/kg).

Flow cytometry and isolation of HSCs. Splenocytes and peripheral blood cells were
incubated with conjugated antibodies and viability exclusion dies. The antibodies used are
listed in Table S2. Stained cells were quantified using a Gallios (Beckman Coulter), and
analyzed with FlowJo software (Treestar). HSCs and LSKs were isolated by depleting Lin+
cells using the Lineage Cell Depletion Kit according to the manufacturer’s protocol (Miltenyi
Biotec), stained with Lin- antibody cocktail, and antibodies against CD117, Sca-1, CD150 and
CD48, and sorted with FACS AriaTM (BD Biosciences).

Cell culture. Lin- cells were cultured in StemSpan medium (StemCell Technologies)
supplemented with 5% FBS, 1% penicillin/streptomycin, recombinant hTPO (100µg/mL),
recombinant mSCF (100µg/mL) and recombinant mFLT3 ligand (100µg/mL). Tunicamycin
(Cayman Chemical) was added (0,6 or 1,2µg/mL) for 24 or 48 hours.

RNA-sequencing. RNA was extracted using the ZR-RNA MicroPrepTM isolation kit
(Proteinegene). cDNA libraries were generated by using Ovation SoLo RNA-seq system
(NuGEN). Control of libraries was performed with the High Sensitivity DNA Analysis Kit
using the Bioanalyzer (Agilent). NextSeq 500 (Illumina) was used for sequencing. FASTQ
files were mapped to the ENSEMBL MM38 reference using Hisat2 and counts were produced
with feature Counts. Read count normalizations and groups comparisons were performed by
DESeq2, edgeR, LimmaVoom. Heatmaps were made with the R and imaged by Java

5
Leveau et al. Ttc7a controls HSC functions…

Treeview software. Differentially expressed genes were examined with GSEA for functional
enrichment in GO terms using normalized expression values of LimmaVoom.

Western blot. Lin- cells were cultured for 3 days and HSCs were sorted directly into 10%
trichloroacetic acid (TCA). Extraction and solubilization of proteins was performed as
previously described 24.

Statistical analysis. Data were analyzed with GraphPad Prism 6 software. Statistical analyses
were performed using two-tailed student t-tests. Differences were considered to be
statistically significant when p < 0,05 (indicated as *p<0,05, **p<0,01, ***p<0,001 and
****p>0,0001).

Data availability. The data are available at the SRA database under the accession number
SRA139913.

6
Leveau et al. Ttc7a controls HSC functions…

Results
Ttc7a is required for the maintenance of immune homeostasis.
It has previously been shown that adult Ttc7a-deficient (fsn) mice (aged 8 to 10 weeks)
develop an imbalance in haematopoiesis, characterized by leukocytosis and anaemia 7. To
gain insight into the change over time in the fsn mice’s pathology, we analysed the different
haematopoietic lineages in the blood and the spleen at 3, 6 and 12 weeks of age. Fsn mice had
a considerably higher circulating leukocyte count than control littermates (ctrl) at all time
points (Fig. 1A). The spleen was much larger in fsn mice than in ctrl mice, twice as large at 3
weeks and 10 times as large at 12 weeks (Fig. 1B). The splenic architecture in fsn mice
became increasingly disorganized, with an age-related expansion of red and white pulps (Fig.
1C). Furthermore, histological assessment of splenic sections revealed extramedullary
haematopoiesis as evidenced by elevated counts of megakaryocytes (Fig. 1C) and of
haematopoietic stem and progenitor cells (HSPCs) (Fig. S1). Relative to ctrl mice, the
absolute splenic T cell count in fsn mice was slightly lower at 3 weeks of age but higher at 6
and 12 weeks of age (Fig. 1D). A large proportion of Ttc7a-deficient T lymphocytes had an
effector memory phenotype (CD44+ CD62L-) (Fig. 1E). Splenic B cell counts were slightly
elevated, and B cells presented the impaired maturation phenotype as previously described in
fsn mice6 (Fig. 1F). The lymphoid alterations were accompanied by massive
myeloproliferation, with an increase over time in the numbers of splenic granulocytes (both
neutrophils and eosinophils) and resident and inflammatory monocytes (Fig. 1G-H). Thus,
Ttc7a-deficient mice displayed a number of persistent haematopoietic alterations (i.e.
leukocytosis, T lymphocyte activation and anaemia) at a very early age, whereas other
manifestations appeared later in life and/or were exacerbated with age (i.e. myeloproliferation
and elevated T cell counts).
Since all the peripheral haematopoietic lineages were affected in fsn mice, we next
looked at whether the HSPC compartment was also altered. In contrast to ctrl mice, bone
marrow (BM) cellularity increased in fsn mice between 3 and 12 weeks of age (Fig. 2A). The
LSK (Lin- Sca1+ cKit+) stem cell population was slightly higher in fsn than in ctrl mice at all
the time points analysed (Fig. S2A). Proportion of HSCs (Lin- Sca1+ cKit+ CD150+ CD48-) 25
were decreased, whereas proportion of more mature haematopoietic progenitor cells (HPC-1:
Lin- Sca1+ cKit+ CD150- CD48+) were increased, compared to ctrl mice (Fig. 2B and Fig.
S2B). At 12 weeks of age, the HSCs progenitor count was significantly lower than the ctrl
value in fsn mice, while the numbers of multipotent progenitors (MPPs: Lin- Sca1+ cKit+
CD150- CD48-) were unchanged and those of HPC-2 (Lin- Sca1+ cKit+ CD150+ CD48+) and

7
Leveau et al. Ttc7a controls HSC functions…

HPC-1 were slightly higher (Fig. 2C). Within the committed progenitor compartment,
numbers of common myeloid progenitors (CMPs: LIN- Sca1- cKit+ CD34+ CD16/32low) and
granulocyte/monocyte progenitors (GMPs: LIN- Sca1- cKit+ CD34+ CD16/32+) were lower
than ctrl values at 3 weeks of age, although the differences disappeared with time (Fig. 2D-E).
There were no significant differences in fsn vs. ctrl values in the numbers of common
lymphoid progenitors (CLPs: LIN- Sca1- cKitint) or megakaryocyte erythrocyte progenitors
(MEPs: LIN- Sca1- cKit+ CD34- CD16/32-) (Fig. 2D-E). We confirmed previous reports
whereby the profound anaemia observed in fsn mice (Fig. S3A) is peripheral in nature and
does not result from a decreased number of early erythroid progenitors but rather from a
defect in the last step of erythropoiesis (Fig. S3B-C) 7. Erythropoiesis and enucleation
26
processes have been shown to involve chromatin compaction and actin cytoskeleton
27
dynamics . Interestingly, we have previously shown that Ttc7a plays a role in actin
15, 16 28
dynamics as well as in chromatin compaction and genomic stability . Hence, it is
tempting to speculate that altered actin dynamics and chromatin organization, as a
consequence of Ttc7a-deficiency, contribute to defective erythrocyte generation in fsn mice.
A high splenic erythroblast count suggested the presence of stress erythropoiesis as a possible
attempt to compensate for the peripheral anaemia (Fig. S3B).
Thus, our present results show that the absence of Ttc7a in fsn mice is associated with
deregulation of the homeostatic balance between haematopoietic lineages, from the stage
HSC onwards, and a tendency of all leukocyte subsets to expand over time.

Ttc7a has an intrinsic role in the fate of progenitor cells.

Since Ttc7a is broadly expressed, it was not possible to distinguish the respective
involvements of haematopoietic factors (i.e. HSCs) and non-hematopoietic factors (e.g. BM
niches and the thymic epithelium) in the generation of the fsn phenotype. In a previous study
we have found that the skin barrier is impaired in fsn mice 9; this defect may enhance antigen
sensitization and thus induce immune system activation. Therefore, in order to determine the
Ttc7a-deficient haematopoietic cells’ intrinsic contribution to fsn-associated haematological
manifestations, we generated chimeric mice by reconstituting lethally irradiated control
recipients with LSK cells purified from either ctrl or fsn mice. Hereafter, these chimeric mice
are respectively referred to as Ctrlctrl and Ctrlfsn. Three-weeks-old mice were chosen as donors
so that we could use a similar LSK graft inoculum in both control and fsn samples, and thus
minimize the potential immune consequences caused by the altered fsn skin barrier. We
monitored the haematological reconstitution over time by collecting blood samples from the

8
Leveau et al. Ttc7a controls HSC functions…

recipient mice, every two weeks. As observed in native fsn mice, white blood cell counts were
higher in Ctrlfsn than in Ctrlctrl mice, and a difference was observed as early as 4 weeks after
transplantation (Fig. 3A). The Ctrlfsn mice were also anaemic (Fig. 3B) and developed
splenomegaly (Fig. 3C), although the latter was less pronounced than in native fsn mice (Fig.
1B). The total body weight of Ctrlctrl and Ctrlfsn mice was not different. The distribution of the
splenic myeloid, T and B cell populations was the same as in ctrl mice (Fig. 3D). As observed
in fsn mice, BM cellularity was higher in Ctrlfsn mice than in Ctrlctrl mice (Fig. 3E), whereas
the LSK counts were slightly increased (Fig. 3F). The distribution of the HSCs, MPPs, HPC-2
and HPC-1 populations was similar in Ctrlfsn and Ctrlctrl mice, suggesting that the low HSC
count observed in 12 weeks-old native fsn mice (Fig. 2C) is primarily caused by external (i.e.
non-haematopoietic) factors. Taken as a whole, these data suggest that Ttc7a has an intrinsic
role in haematopoietic cells; the absence of Ttc7a in haematopoietic progenitors results in the
over-proliferation of the various cell lineages as seen in native fsn mice.

Loss of Ttc7a enhances the reconstitution potential of HSC.

Next, we sought to determine the impact of Ttc7a loss on the HSCs’ reconstitution potential
in a controlled in vivo environment. Using lethally irradiated congenic recipients, we
transferred equal numbers of LSK cells purified from 3-weeks-old ctrl (LSKctrl)- or fsn
(LSKfsn)-(CD45.2+) mice together with competitor wild-type-(CD45.1+) BM cells (i.e. Ctrl-
LSKctrl or Ctrl-LSKfsn). We then assessed the respective contributions of cells originating
from ctrl or fsn LSK donors during haematopoietic reconstitution. As early as 2 weeks post-
transfer, the proportion of LSKfsn donor-derived leukocytes in the recipients’ blood was
higher than that of LSKctrl. These differences persisted 14 weeks after transfer (Fig. 4A). The
proportion of cells originating from donor LSK in the recipients’ organs, and particularly the
thymus and the BM, was higher in Ctrl-LSKfsn mice than in Ctrl-LSKctrl mice (Fig. 4B). In the
spleen, the reconstitution advantage of LSKfsn donor-derived cells led to the expansion of
neutrophil, eosinophil and monocyte lineages and to a lesser extent, T cell lineages (Fig. 4C).
To further evaluate the effect of Ttc7a-loss on long-term reconstitution, total BM cells from
primary recipient mice were transplanted into secondary recipients. The competitive
advantage of Ttc7a-deficient LSK donor cells with regards to reconstitution was maintained
and even enhanced upon secondary and tertiary transplantation (Fig. 4D). Thus, our results
show that a defect in Ttc7a improves the competitive fitness of HSCs following
transplantation.

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Leveau et al. Ttc7a controls HSC functions…

Loss of Ttc7a increases the long-term self-renewal potential of HSCs.

In view of the elevated proliferative capacity of Ttc7a-deficient haematopoietic cells, we next
sought to assess HSCs’ properties that could modify their reconstitution potential (i.e.
quiescence and self-renewal capacity). To evaluate the impact of Ttc7a loss on the quiescence
of the reconstituting HSCs, we measured BrdU incorporation in control and Ttc7a-deficient
HSPCs before and after bone marrow transplantation. Upon 24 hours of BrdU treatment, we
observed similar percentages of BrdU positive (BrdU+) HSC, HPC-1 and HPC-2 cells
purified from control and fsn mice (Fig. S4A). Similar results were obtained when comparing
cell cycle progression in control and Ttc7a-deficient HSCs upon transplantation of irradiated
recipients (Fig S4B-C). Altogether, these results suggest that the increased repopulation
capacity of fsn HSCs was not caused by a disturbed quiescent state.
We then looked at whether the HSCs’ long-term self-renewal ability was altered in this
context. We therefore performed serial BM transplantations from irradiated mice having
received whole BM from 3-weeks-old ctrl or fsn mice. Unexpectedly, Ttc7a-deficient cells
successfully sustained BM reconstitution longer than ctrl cells did. The ctrl HSCs sustained 6
rounds of transplantation (Fig. 5A) but all the Ctrlctrl mice died during the 7th round of BM
transfer (Fig. 5A-B). In contrast, most Ctrlfsn mice survived 6 weeks after the 7th round and
were able to undergo an additional round of transplantation before dying after the 8th round
(Fig. 5A-B). Similar results were obtained in experiments with 12-weeks-old donors (Fig.
S5A-B). We next determined the ability of ctrl and Ttc7a-deficient BM cells from 3-weeks-
old mice to properly reconstitute haematopoiesis over the different rounds of transplantation.
During the first five rounds, the same phenotype was always observed. Ctrlfsn mice displayed
anaemia (Fig. 5C), together with an elevated leukocyte count up until the 3rd round (Fig. 5D).
The spleen size was larger in Ctrlfsn mice than in Ctrlctrl mice until the 5th round (Fig. 5E).
However, differences between Ctrlfsn and Ctrlctrl mice were no longer observed in the 6th
round; this was probably caused by exhausted donor cells that failed to properly reconstitute
recipient mice at the end of the reconstitution process (Fig. 5C-E). Interestingly, the
distribution of splenic leukocyte subsets in the Ctrlfsn mice was progressively biased toward
myeloid populations at the expense of B cells, and to a lesser extent, T cells, as notably
observed for the 5th round (Fig. 5F). In Ctrlfsn mice, this bias became detectable in the 3rd
round and persisted until the 7th round (Fig. S5C). In summary, these data show that Ttc7a-
deficient HSCs have a greater ability to self-renew and to induce myeloid cell expansion.

10
Leveau et al. Ttc7a controls HSC functions…

Ttc7a-deficiency perturbs the transcriptomic profile of ER stress response in HSCs.

In order to gain further mechanistic insight into the Ttc7a-related regulation of HSC
homeostasis, we carried out a transcriptomic analysis of HSCs isolated from Ttc7a-deficient
and control BM (from 4-weeks-old mice). A two-way hierarchical clustering analysis of
differentially expressed genes (DEGs) (P value ≤0,05, fold change≥1,2) revealed a clear-cut
separation between Ttc7a-deficient and control HSCs samples (Fig. 6A). We found that 1103
genes were significantly upregulated and 928 significantly downregulated in Ttc7a-deficient
HSCs relative to the expression levels in control HSCs (Fig. 6B). Among the DEGs, the most
statistically significant differences were observed in the group of the down-regulated genes
(Fig. 6B). To determine the DEGs’ functional profile, we performed a gene set enrichment
analysis (GSEA) for genes with a fold change ≥1.5. A gene ontology analysis of the identified
gene signature revealed a significant enrichment of genes in three main categories. Two
categories are related to chromatin organization/modification and DNA damage repair; this
observation fits with our recent finding that Ttc7a is a chromatin-binding nuclear factor
involved in chromatin compaction and nuclear organization 28 (Fig. 6C-D). Another category
corresponds to genes involved in cellular response to stress (Fig. 6E). Our transcriptomic
analysis also highlighted high expression levels of Ttc7a in HSCs (Fig. S6). A growing body
of evidence suggests that ER stress regulates the function of the HSC pool 21. In particular, a
recent study highlighted a link between ER stress perturbation in HSCs and an elevated
29
reconstitution capacity following BM transplantation . Accordingly, we found that several
effectors of the ER stress response were significantly downregulated in Ttc7a-deficient HSCs,
including the unfolded protein response (UPR) master regulator Bip (Hspa5/GRP78),
calreticulin, Pdia3, Pdia4, Pdia6, several heat shock proteins (Hsps), as well as Sel1l and
Syvn1 which have been shown to regulate ER-associated protein degradation (ERAD)
30, 31
pathway (Fig. 6F and Table S1). Overall, our data suggest that Ttc7a loss impacts the
cellular response to ER stress in HSCs.

Ttc7a controls the response to stress in HSCs.

ER stress is mainly triggered in response to altered protein homeostasis leading to pro-
apoptotic or pro-survival responses. Notably, Ttc7a-deficient HSCs presented with reduced
levels of protein aggregation compared to control HSCs (Fig. 7A). In keeping with this, in
vitro expanded fsn HSCs presented with elevated BIP protein level (Fig. 7B). These results

11
Leveau et al. Ttc7a controls HSC functions…

suggest that Ttc7a loss could modify HSCs susceptibility to ER stress. Therefore, to
determine the impact of Ttc7a loss in HSCs’ response to ER stress, we analysed the
proliferative capacity of Ttc7a-deficient HSCs and progenitor cells (i.e. HSC, MPP, HPC-2
and HPC-1) upon chemical induction of ER stress in vitro. To do so, lineage-negative cells
from 4-weeks-old fsn and ctrl mice were cultured for 48 hours in the presence or absence of
Tunicamycin (TM). TM blocks the synthesis of N-linked glycoproteins, leading to an
accumulation of unfolded proteins and the induction of ER stress 32. As expected, on day 2,
TM treatment reduced the proliferation ability of control cells in a dose-dependent manner
(Fig. 7C). In contrast, at a low dose of TM, the proliferative capacity of Ttc7a-deficient HSCs
was significantly higher than that of control HSCs (Fig. 7C). These differences were
particular to HSCs, as Ttc7a-deficient MPP, HPC-2 and HPC-1 subsets presented with a
similar response to TM than their control counterparts (Fig. S7A). The alterations in the ER
stress response in Ttc7a-deficient HSCs were not due to protein aggregation (Fig. 7B and
S7B), nor low expression of the ER stress sensors and effectors (Ire1α, Perk, Atf6, etc), as no
differences were observed in our transcriptomic analysis (Fig. S7C and Table S1).
Surprisingly, the reduction in cell proliferation of ctrl HSCs in response to TM was
independent of apoptosis, in contrast to other progenitor cells. Apoptosis of Ttc7a-deficient
HSCs was reduced compared to unstimulated ctrl cells, and remained unchanged upon TM
treatment (Fig S7D). No differences were observed in other progenitor populations (Fig.
S7D). Altogether, these data suggest that ex vivo purified Ttc7a-deficient HSCs presented
with a higher level of ER stress compared to their control counterparts.
Interestingly, we observed that Hsp70, a chaperone associated to broad cellular stresses, also
presented an increased protein expression in fsn HSCs compared to controls (Fig S7E). In
order to determine whether Ttc7a regulates the cellular response to stress in vivo, we
monitored the proliferative response of Ttc7a-deficient cells following the induction of stress
by 5-fluorouracile (5-FU). The depletion of cycling cells by 5-FU stimulates HSCs to
replenish peripheral leukocytes 33, 34, inducing a broad stress response in HSCs, not limited to
ER stress (e.g. oxidative stress, proliferative stress). We injected 5-FU into Ctrlfsn and Ctrlctrl
mice 3 months after BM transplantation and monitored the replenishment of peripheral
leukocytes for 19 days. The Ttc7a-deficient and ctrl leukocyte counts fell until day 9 post-
injection, and then increased. On day 15, Ttc7a-deficient leukocytes were growing
significantly more strongly than ctrl cells, with a peak on day 16 (Fig. 7D). Interestingly, the
spleen of Ctrlfsn mice enlarged further after 5-FU treatment (Fig. 7E, compared with Fig. 3D),
with higher lymphoid and myeloid counts (Fig. 7F-G). To assess the proliferative response of

12
Leveau et al. Ttc7a controls HSC functions…

Ttc7a-deficient HSCs following stress injury, we assayed bromodeoxyuridine (BrdU) uptake

by LSK subsets between day 6 and day 7 after 5-FU injection. The greater BrdU uptake in
Ttc7a-deficient HSCs, MPPs, HPC-2 and HPC-1 (Fig. 7H), suggested that Ttc7a controls the
cell cycle progression of HSCs under stress conditions. Strikingly, BrdU uptake did not differ
in committed progenitors, CLPs, CMPs, GMPs and MEPs (Fig. 7I). These results suggest that
Ttc7a is involved in the regulation of HSCs proliferative response under stress conditions but
not in that of committed progenitor cells.

13
Leveau et al. Ttc7a controls HSC functions…

Discussion
The present study revealed a previously unrecognized role for Ttc7a in the negative regulation
of HSCs function. Using murine transplantation models and Ttc7a-deficient HSCs, we found
that Ttc7a intrinsically regulates the maintenance and proliferation of HSCs in vivo, and the
subsequent homeostasis of downstream cell populations. We also found that Ttc7a expression
in HSCs is closely associated with the transcriptional response to ER stress.
Hematopoietic stem cells are the only cells capable to self-renew and differentiate into
all mature blood lineages. The quiescence of HSCs must be tightly regulated in order to
35, 36
control proliferation, maintain normal homeostasis, and prevent stem cell exhaustion .
Various intrinsic and cell-extrinsic regulatory factors of HSCs’ cell cycle have been
described, such as phosphatase and tensin homologue (Pten) signaling, Wnt signaling and
cytokine signalling 37. Indeed, in mice that lack growth factor independent 1 (Gfi1) 38, Pten,
39 20, 36
forkhead box proteins 1, 3, 4, or M1 or other proteins , excessive HSC proliferation is
associated with stem cell exhaustion and the loss of self-renewal. In contrast, we found that
mice reconstituted with Ttc7a-deficient progenitors exhibit a characteristic phenotype with an
enhanced HSC function, higher HSC-derived peripheral blood cell counts and no evidence of
stem cell exhaustion when compared with Ttc7a-proficient HSCs. Indeed, Ttc7a-deficient
HSCs were better able to repopulate the haematopoietic system in serial transplantation
experiments, indicating that the self-renewal of Ttc7a-deficient HSCs is not compromised by
repeated rounds of proliferation. Although this situation clearly differs from the above-
mentioned knock-out mice, a few similar observations have been reported after the deletion of
40
the cyclin-dependent kinase 4 inhibitor C (CDKN2C) , the ubiquitin-mediated protein
degradation Cbl 41 and Itch 41, and the transcription factors Hif1a 42
and Egr1 43. The loss of
these proteins enabled the maintenance of HSCs, despite an increased in their proliferative
capacity. However, the specific mechanisms by which these proteins regulate HSC function
mostly remain unknown.
Our findings support a role for the ER stress response in the enhanced function of
Ttc7a-deficient HSCs. Since long-lived HSCs are particularly sensitive to stress stimuli, their
response must be tightly controlled in order to prevent either a loss of function or the clonal
persistence of oncogenic mutations. It has been shown that HSCs are enriched in components
of the UPR pathway. Upon exposure to acute stress in vitro, HSCs are more prone to
apoptosis, via upregulation of the canonical UPR genes, than related progenitors having lost
21, 44
their self-renewal capacity . Along these lines, the ectopic expression of developmental
pluripotency-associated 5 (Dppa5) was associated with enhanced HSC function, via

14
Leveau et al. Ttc7a controls HSC functions…

suppression of the ER stress response (by downregulating the expression of ER stress

chaperones) and the subsequent apoptotic signals 29. However, UPR activation can also have
an anti-apoptotic outcome in HSCs. It has been shown that stimulation of the estrogen
receptor α (ERα) confers HSCs resistance to proteotoxic stress by activating the Ire1α-Xbp1
24
branch of the UPR and promoting their reconstitution potential . In Ttc7a-deficient HSCs,
expression levels of ER stress response genes were abnormally low, whereas protein level of
Bip chaperonne was increased. Furthermore, Ttc7a-deficient HSCs were less sensitive to
stress induction than Ttc7a-proficient-HSCs both in vitro and in vivo. A similar phenomenon
was observed in mouse liver where mild chronic ER stress decreases mRNA level of Bip
while maintaining its protein level. This response allows hepatocytes to avoid the
45
overproduction of UPR effectors that could lead to apoptosis . Therefore, it is tempting to
speculate that Ttc7a-deficiency could be associated with mild chronic stress. In this context,
the increased resistance of fsn HSCs to tunicamycin, could be caused by a cellular adaptive
response aiming to increase the threshold of ER stress sensitivity, and ensure cell survival.
Interestingly, HSCs exposed to other sources of persistent cellular stress develop mechanisms
of stress resistance resulting an increased self-renewal capacity and reconstitution potential 46.
Knowing that TTC7A stabilizes several interacting proteins (2), the role of additional
components, altered as a consequence of Ttc7a-deficiency, cannot be excluded, Ttc7a could
represent a pivotal connection between ER stress regulation and the maintenance of HSCs
functions.
Along with an abnormally proliferative hematopoietic system, fsn mice develop
hyperplasia of the epidermis and the gastric epithelium. Notably, stem cells from other tissues
can similarly sense ER stress and activate the UPR pathway to control self-renewal and
differentiation. This has been shown for the intestinal epithelium in particular, and several
lines of evidences support the concept whereby ER stress and UPR activity regulate the
47
differentiation of intestinal stem cells . An attractive hypothesis would be that the other
phenotypic manifestations that characterized Ttc7a-deficiency might be due to perturbation of
the ER stress response.
In summary, our results show that Ttc7a has a critical but previously unrecognized
role as a regulator of HSC homeostasis and function through the regulation of ER stress
response.

15
Leveau et al. Ttc7a controls HSC functions…

Acknowledgements:
We thank Gaël Ménasché, Annarita Miccio and Isabelle Andre-Schmutz for helpful
comments and guidance; Olivier Pellé for help in cell sorting; The Necker histology and
morphology facility (Structure Fédérative de Recherche (SFR) Necker) and the Cochin
genomic Platform for their service in histological and transcriptomic studies respectively.
This work was supported by The French National Institutes of Health and Medical Research
(INSERM), the state funding from the Agence Nationale de la Recherche “Investissements
d’avenir” program, la Fondation pour la Recherche Médicale (FRM project
DEQ20150734354), and the Imagine Foundation. C.L. was supported by a fellowship from
the Ministry of Education and FRM. M.T.E.D by fellowship from the ANR, the FRM and the
European Research council (ERC). T.G. is a fellow of the International PhD. program of the
Imagine Institute funded by the Bettencourt Schueller Foundation.

Contributions
C.L. designed and performed experiments, analysed data and wrote the paper. T.G. performed
experiments and analysed data. M.T.E.D. participated to data analysis and the discussions
along the project. N.C. conducted bioinformatics analysis. A.F. participated to the discussions
along the project and edited the manuscript. G.d.S.B. and F.E.S. conceived the study,
analysed data, wrote the manuscript and supervised the overall research.

Conflict of interest
The authors declare that they have no conflict of interest.

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Leveau et al. Ttc7a controls HSC functions…

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Figure legends:

Figure 1 - Ttc7a-deficiency perturbs homeostasis of all immune populations. Control

littermates (ctrl – black bars) and Ttc7a-deficient (fsn – red bars) mice were analysed at 3, 6
and 12 weeks of age (mean ± SEM) *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001 (two-
tailed t-test). (A) White blood cell count (n≥6). (B) Spleen size determined as percent of body
weight (n≥6). (C) Histological sections of spleen stained with hematoxylin and eosin (HE)
showing megakaryocytes (black arrow) (left panel) and quantification of spleen
megakaryocytes (right panel). (D) Total number of T cells in the spleen (n≥7). (E) Flow
cytometry representative of T cell activation (according to CD44 and CD62L expression) at
12 weeks (left panel), and activation index of T cells (ratio of effector memory T cells
(CD44+ CD62L-) over naive T cells (CD44- CD62L+)) (right panel) of fsn and ctrl mice (n≥7).
Numbers adjacent of outlined areas indicate percent cells in parent gate (mean). (F) Total
number of B cells in the spleen (n=4) (left panel) and flow cytometry representative of B cell
maturation (according to IgM and IgD expression) (right panel). (G) Representative flow
cytometry at 12 weeks (left panel) and total number of neutrophils (CD11b+ Ly6Ghi) and
eosinophils (CD11b+ Ly6Gint SSChi). (H) Total number of inflammatory (CD11b+ Ly6G-
Ly6C+) and resident monocytes (CD11b+ Ly6G- Ly6C-) (n≥6). Numbers adjacent of outlined
areas indicate percent cells among leukocytes in the spleen (mean).

Figure 2 – Ttc7a-deficiency alters the HSPC compartment. HSPC compartment was

analysed in the bone marrow (BM) of 3, 6 and 12 weeks old ctrl (black bars) and Ttc7a-
deficient (fsn - red bars) mice (mean ± SEM) *P<0,05; **P<0,01; ****P<0,0001 (two-tailed
t-test). (A) Quantification of total femur BM cells (n≥6). (B) Representative flow cytometry at
12 weeks (left panel) and percentage of HSC (Lin- Sca1+ cKit+ CD150+ CD48-) among LSK
cells (right panel) (n≥7). (C) Quantification of LSK cell populations, HSC, MPP (Lin- Sca1+
cKit+ CD150- CD48-), HPC-2 (Lin- Sca1+ cKit+ CD150+ CD48+) and HPC-1 (Lin- Sca1+ cKit+
CD150- CD48+). (D) Representative flow cytometry at 12 weeks and (E) quantification of
CLP (Lin- Sca1- cKitint CD127+), CMP (Lin- Sca1- cKit+ CD34+ CD16/32-), GMP (Lin- Sca1-
cKit+ CD34+ CD16/32+) and MEP (Lin- Sca1- cKit+ CD34- CD16/32-) (n≥7). (B-D) Numbers
adjacent of outlined areas indicate percent cells in parent gate (mean). Numbers in parenthesis
indicate percentage among leucokytes in the bone marrow (mean).

20
Leveau et al. Ttc7a controls HSC functions…

Figure 3 – Transferred Ttc7a-deficient LSKs reproduce fsn manifestations in a control

environment. Control irradiated mice were transferred with LSK cells purified from 3 weeks
old ctrl (Ctrlctrl – black bars) or Ttc7a-deficient mice (Ctrlfsn – red bars). (mean ± SEM)
*P<0,05; **P<0,01 (two-tailed t-test). (A) Monitoring of the number of leukocytes and (B)
red blood cells, hematocrit and hemoglobin during hematopoietic reconstitution. (C) Spleen
size determined as percent of body weight 10 weeks after BM transfer. (D) Relative
contribution of myeloid compared to T cells and B cells in the spleen. (E) BM cellularity and
(F) absolute number of LSK cells 10 weeks after BM transfer.

Figure 4 - Ttc7a-deficient HSCs exhibit a higher repopulation capacity. (A-C) Lethally

irradiated CD45.1 mice were reconstituted with a mix of control whole BM and sorted LSK
cells purified from ctrl (Ctrl-LSKctrl – black bars and lines) or Ttc7a-deficient mice (Ctrl-
LSKfsn – red bars and lines) (mean ± SEM) *P<0,05; **P<0,01; ***P<0,001; ****P<0,0001
(two-tailed t-test). These data are representative of three independent experiments. Proportion
of LSK donor-derived leukocytes (A) in the blood over time, (B) in lymphoid organs (15
weeks after transfer) and (C) in the spleen for the different types of leukocytes (n=8). (D)
Bone marrow cells from 1st and then 2nd round recipient mice of each group were pulled and
transplanted to secondary and tertiary ctrl CD45.1 recipients respectively. Proportion of LSK
donor-derived leukocytes of Ctrl-LSKctrl and Ctrl-LSKfsn was determined in each round (n=14
for control and n=16 for Ttc7a-reconstituted mice).

Figure 5 – Ttc7a-deficiency promotes self-renewal ability of HSC. Lethally irradiated mice

were serially transplanted with 3 weeks old ctrl (Ctrlctrl – black bars, dots and lines) or Ttc7a-
deficient (Ctrlfsn – red bars, dots and lines) donor BM cells (mean ± SEM) *P<0,05;
**P<0,01; ***P<0,001; ****P<0,0001 (two-tailed t-test). These data are representative of 4
independent experiments with at least 4 mice per each round of transplantation (A) Percent
survival of recipient mice across the 7 transplantation cycles. (B) Survival of Ctrlctrl and Ctrlfsn
mice during the 7th round and Ctrlfsn mice during the 8th over time (n=4 for control- and n=10
for Ttc7a-reconstituted mice). (C) Red blood cells, hematocrit, hemoglobin, (D) leukocytes
count and (E) spleen weight across the transplantation cycles. (F) Relative contribution of
myeloid compared to T and B lymphoid cells in the spleen of mice transferred with BM cells
that had undergone 5 transplantation cycles.

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Leveau et al. Ttc7a controls HSC functions…

Figure 6 – Ttc7a-deficiency results in a reduced expression of ER stress response genes

in HSCs. RNA sequencing was performed on 3 weeks old control (ctrl – black bars) and
Ttc7a-deficient (fsn – red bars) HSCs transcripts. (A) Heatmap of relative expression of DEGs
(fold change≥1,2) in Ttc7a-deficient HSCs compared to control. (B) Volcanoplot of DEGs
(fold change ≥1,2) in Ttc7a-deficient HSCs compared to control showing the adjusted p-value
(-log10) versus fold change (log2). Up-regulated and down-regulated genes are shown in red
and blue respectively. Total numbers in each group are indicated in red and blue respectively.
(C-E) Enriched gene sets in Ttc7a-deficient HSCs compared to control, as determined by
GSEA of DEGs (fold change ≥1,5). (F) Normalized expression of ER stress response genes
down-regulated in Ttc7a-deficient HSCs (fold change ≥1,2) *P<0,05; **P<0,01;
****P<0,0001 (LimmaVoom analysis).

Figure 7 –Ttc7a controls the response to stress in HSCs. (A) Representative histograms of
protein aggregation level of 3 weeks old ctrl (black line) and fsn (red line) HSCs. (B) Protein
expression of Bip in ctrl and fsn HSCs after 3 days in vitro expansion. (C) Proliferation index
(calculated as the ratio between the number of cells at 48 hours and 24 hours) of HSCs after
Lin- cells were sorted from ctrl (black bars) and Ttc7a-deficient (fsn – red bars) mice and
cultured for two days with or without tunicamycin (TM). **p<0,01 (two-tailed t-test). (D-I)
Ctrlctrl (black line and bars) and Ctrlfsn (red line and bars) mice were analysed after they
received a single intraperitoneal injection of 150mg/Kg of 5-FU, 12 weeks after BM transfer.
(D) White blood cell count over time. (E) Spleen size, (F) absolute number of lymphoid and
(G) myeloid cells in the spleen 15 days after 5-FU injection (n=9). (H) BrdU incorporation of
LSK subpopulations and (I) representative flow cytometry histogram of LK populations of
BrdU incorporation 7 days after 5-FU injection. (n=10 for control- and n=12 for Ttc7a-
reconstituted mice) *P<0,05; **P<0,01; ***P<0,001 (two-tailed t-test).

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Leveau et al. Ttc7a controls HSC functions…

Supplementary Methods
Mice. Heterozygous Balb/cByJ fsn (CByJ.A-Ttc7fsn/J) mice and Balb/cByJ CD45.1
(CByJ.SJL(B6)-Ptprca/J) mice were obtained from the Jackson Laboratory.
Heterozygous Ttc7afsn/+ mice were bred to generate homozygous Ttc7afsn/fsn mice and
their respective control littermates. Genotyping of Ttc7a gene was performed by PCR
using the following primers: Ttc7a_F: 5’-CTCTTTCCATGGCTTCCTTG-3’,
Ttc7awt_R: 5’-GAAGAGCAGCGCTAGCAAGT-3’, Ttc7afsn_R: 5’-
TAGAAAGGTGCACGGGTGTG-3’.

Repopulations assays. For bone marrow transplantation experiments, CD45.1+ ctrl

recipient mice were subjected to a single dose of 8 Gray total body irradiation with a X-
ray electric generator RS2000 (RAD SOURCE), and cells were transferred intravenously
in the tail vein. After transplantation, peripheral blood was sampled every two weeks to
monitor cell engraftment. Blood counts were determined by ScilVet Abc+ analysis. Bone
marrow reconstitution with LSK donors was performed by injecting 30 000 cells into
irradiated recipient mice. For serial transplantations, primary recipients were
reconstituted with 107 cells as described above. Bone marrow cells of primary recipients
were pulled and 107 cells were transferred to secondary recipients. To perform
competitive repopulation assays, 1 000 LSK cells were injected concomitantly with 2 x
106 unfractionated CD45.1+ bone marrow cells.
For 5-FU (5-fluorouracil) treatment, control recipients were reconstituted with 107 control
or Ttc7a-deficient bone marrow cells. 12 weeks after transfer, mice were treated with a
single dose of 5-FU (150 mg/kg intra peritoneally). Peripheral blood was sampled every
3 days to monitor blood cell counts.

Flow cytometry and isolation of HSCs. Cell suspensions were prepared by mechanical
disruption of mouse bone marrow and spleen in PBS. For FACS analysis of spleen and
peripheral blood, red blood cells were lysed with RBC lysis buffer (Biolegend, San Diego,
CA, US). For erythroid differentiation studies, FACS analyses were performed without
RBC lysis. Dead cells were stained with LIVE/DEADTM Fixable Aqua Stain
(ThermoFischer Scientific, Watham, MA, US), 20 minutes at 4°C. Cells were then
incubated with conjugated antibodies at the appropriated concentration for 20 minutes at
4°C in darkness. For Annexin V staining, cells were incubated 15 minutes at room
temperature with Annexin V (Sony Biotech, San Jose, CA, US)) and 7-AAD (BD
Leveau et al. Ttc7a controls HSC functions…

Pharmingen) in Annexin V binding buffer (Biolegend). The antibodies used are listed in
Table S2. Stained cells were quantified using a Gallios (Beckman Coulter), and FlowJo
software (Treestar) was used to generate flow cytometry plots and histograms. LSKs were
isolated by depleting Lin+ cells using the Lineage Cell Depletion Kit according to the
manufacturer’s protocol (Miltenyi Biotec). The enriched cells were stained with Lin-
antibody cocktail, and antibodies against CD117 (c-Kit), Sca-1 (Ly6A/E), CD150
(Slamf1) and CD48 (Slamf2), and sorted with FACS AriaTM (BD Biosciences). For BrdU
incorporation assay, control and Ttc7a-deficient mice received a single i.p. injection of
BrdU. 24 hours later bone marrow cells were harvested and analyzed for BrdU
incorporation using the BrdU Flow kit according to manufacturer’s instructions (BD
Biosciences), associated to viability staining and surface staining to define BM subsets.
For cell cycle analysis, Lin- cells were stained with Ki67 antibody and Hoechst 33342
with Intracellular Fixation and Permeabilization Buffer set according to manufacturer’s
protocol (Ebiosciences). For aggresome detection, Lin- cells were stained using the
Aggresome detection kit according to the manufacturer’s protocol (Abcam, Cambridge,
UK).

Cell culture. Bone marrow cells were harvested and lineage depletion was performed
using Lineage Cell Depletion Kit according to the manufacturer’s protocol (Miltenyi
Biotec). 250,000 Lin- cells were plated at 106/mL in StemSpan medium (StemCell
Technologies) supplemented with 5% FBS, 1% penicillin/streptomycin, recombinant
human TPO (100 µg/mL), recombinant mouse SCF (100 µg/mL) and recombinant mouse
FLT3 ligand (100 µg/mL). Tunicamycin (Cayman Chemical) was dissolved in DMSO
and added (0,6 or 1,2 µg/mL) for 24 or 48 hours. Cells were counted with Countess™ II
Automated Cell Counter (ThermoFisher Scientific).

RNA-sequencing. HSCs (CD150+ CD48- LSK) were directly sorted into RNA Lysis
Buffer, and RNAs were extracted using the ZR RNA MicroPrepTM isolation kit (Zymo
Research) according to the manufacturer’s protocol. cDNA libraries were generated by
using Ovation SoLo RNA-seq system (NuGEN). Control of libraries was performed with
the High Sensitivity DNA Analysis Kit using the Bioanalyzer (Agilent). NextSeq 500
(Illumina) was used for sequencing with paired-ended sequencing length of 2x75 bases.
FASTQ files were mapped to the ENSEMBL MM38 reference using Hisat2 and counts
were produced with feature Counts. Read count normalizations and groups comparisons
Leveau et al. Ttc7a controls HSC functions…

were performed by three independent and complementary methods – DESeq2, edgeR,

LimmaVoom – and the results were compared and merged. The results were filtered at p
value £0,05 and folds 1,2. Heatmaps were made with the R package ctc: Cluster and tree
Conversion, using normalized expression values of LimmaVoom and imaged by Java
Treeview software. Differentially expressed genes were examined with GSEA for
functional enrichment in GO terms using normalized expression values of LimmaVoom.

Western blot. Lin- cells were cultured for 3 days and HSCs were sorted by FACS directly
into trichloroacetic acid (TCA) and adjusted to a final concentration of 10% TCA.
24
Extraction and solubilization of proteins was performed as previously described .
Proteins were separated on a 4-12% Bis-tris gel (Invitrogen) and transferred to a PVDF
membrane (Millipore). Bip, HSP70 and Gapdh proteins were revealed by western blot
using specific antibodies (Cell Signaling Technology, Millipore, Burlington, MA, US and
Abcam, Cambridge, UK).
Leveau et al. Ttc7a controls HSC functions…

Supplementary Figure legends:

Figure S1 – Total number of HSPCs in the spleen of ctrl (black bars) and fsn (red bars)
mice at 3, 6 and 12 wees of age (n³6) (mean ± SEM). *P<0,05; **P<0,01; ***P<0,001
(two-tailed t-test).

Figure S2 - HSPC compartment was analysed in the bone marrow (BM) of 3, 6 and 12
weeks old ctrl (black bars) and Ttc7a-deficient (fsn - red bars) mice (mean ± SEM)
****P<0,0001 (two-tailed t-test). (A) Total number of LSK cells (n³7). (B) Frequency
of MPP, HPC-2 and HPC-1 among LSK cells (n³6).

Figure S3 – (A) Red blood cell count, hemoglobin and hematocrit of control littermates
(ctrl – black bars) and Ttc7a-deficient (fsn – red bars) mice at 3, 6 and 12 weeks of age
(mean ± SEM) (n³6). (B) Total number of erythroblasts in bone marrow (left panel) and
spleen (right panel) of 12 weeks old ctrl and fsn mice. (C) Flow cytometry representative
of erythroid differentiation (according to CD71 expression and forward scatter) at 12
weeks in fsn and ctrl mice. Numbers adjacent of outlined areas indicate percent cells in
parent gate (mean).*P<0,05; **P<0,01; ***P<0,001; ****P<0,0001 (two-tailed t-test).

Figure S4 – (A) BM cells were collected from ctrl (black dots) and fsn (red dots) mice at
3 and 12 weeks of age, 24 hours after a single injection of BrdU. Percentage of BrdU
positive cells was evaluated in HSC, HPC-2 and HPC-1 by flow cytometry. (B-C) BM
cells were collected from Ctrlctrl (black dots) and Ctrlfsn (red dots) mice 3 weeks after BM
transfer and Ki67/Hoeschst 33342 staining was performed. (B) Percentage of HSCs in
each cell cycle phase was evaluated. (C) Flow cytometry representative of Ki67/Hoechst
33342 staining of HSCs.

Figure S5 – (A-B) Lethally irradiated mice were serially transplanted with 12 weeks old
ctrl (black bars and lines) or Ttc7a-deficient (fsn – red bars and lines) donor BM cells.
These data are representative of 2 independent experiments with at least 3 mice per each
round of transplantation (A) Percent survival of recipient mice across the 7 transplantation
cycles. (B) Survival of Ctrlctrl and Ctrlfsn mice during the 6th round and Ctrlfsn mice during
the 7th over time (n=5 for control- and n=11 for Ttc7a-reconstituted mice). (C) Relative
Leveau et al. Ttc7a controls HSC functions…

contribution of myeloid compared to T and B lymphoid cells in the spleen of irradiated

recipients serially transplanted with 3 weeks old ctrl (black dots) or fsn (red dots) donor
BM cells.

Figure S6 - RNA sequencing was performed on 3 weeks old control (ctrl) LT-HSCs
transcripts. Expression of Ttc7a mRNA transcript was compared to Sca-1, cKit, CD150
and CD48 mRNA transcripts.

Figure S7 – (A) Proliferation index (calculated as the ratio between the number of cells
at 48 hours and 24 hours) of MPP, HPC-2 and HPC-1 after Lin- cells were sorted from
ctrl (black bars) and Ttc7a-deficient (fsn – red bars) mice and cultured for two days with
or without tunicamycin (TM). (B) Representative histograms of protein aggregation level
of ctrl (black line) and fsn (red line) HSCs after 3 days in vitro expansion. Representative
from 3 independent experiments (n=3). (C) Normalized expression of ER stress receptors
and effectors in ctrl and Ttc7a-deficient HSCs (LimmaVoom analysis). (D) Annexin V
staining of HSCs, HPC-1 and HPC-2 after Lin- cells were sorted from ctrl (black bars)
and Ttc7a-deficient (fsn – red bars) mice and cultured for two days with or without
tunicamycin (TM). *p<0,05 (two-tailed t-test). (E) Protein expression of Hsp70 in ctrl
and fsn HSCs after 3 days in vitro expansion (representative of two independent
experiments).
 Table S1 - List of DEGs related to ER stress (p≤0,05; 0,833≥fold change≥1,200)

Sondes Gene RatiosMoys_Fsn_vs_WT Delta pval Limmavoom pval edgeR pval DEseq2
ENSMUSG00000090877 Hspa1b 0.08802294 53.81062 1,54969E-19 1,6719E-15 3,18277E-09
ENSMUSG00000029657 Hsph1 0.40476508 320.17351 2,23955E-27 1,44893E-13 2,15459E-08
ENSMUSG00000026864 Hspa5 0.53204545 890.45856 4,07427E-32 1,71998E-08 4,20833E-08
ENSMUSG00000015656 Hspa8 0.59971221 1105.28708 3,09075E-28 5,30123E-06 1,60832E-07
ENSMUSG00000032575 Manf 0.60433485 82.48086 2,91807E-06 0,000735798 0,002012401
ENSMUSG00000032553 Srprb 0.60691657 8.599529 0,004331196 0,03258362 NS
ENSMUSG00000028410 Dnaja1 0.61410102 353.97319 2,45433E-12 2,57138E-05 5,39726E-05
ENSMUSG00000005374 Tbl2 0.64061516 47.77602 0,001385277 0,01300467 0,02770783
ENSMUSG00000004460 Dnajb11 0.64292208 64.18646 0,00020224 0,004711647 0,01021892
ENSMUSG00000005078 Jkamp 0.65123690 10.030755 0,009853081 0,03230204 NS
ENSMUSG00000005483 Dnajb1 0.65521199 125.18250 5,97843E-06 0,001307077 0,001630244
ENSMUSG00000002835 Chaf1a 0.69009974 39.26364 0,004823073 0,03352666 0,04348155
ENSMUSG00000025198 Erlin1 0.69583222 58.06665 0,000898242 0,01672315 0,02648279
ENSMUSG00000070426 Rnf121 0.70942962 6.9890015 0,01416127 NS NS
ENSMUSG00000057789 Bak1 0.71608208 12.957568 0,005523162 0,0408372 NS
ENSMUSG00000031770 Herpud1 0.71698985 87.60374 0,000628255 0,01306436 0,02067868
ENSMUSG00000035890 Rnf126 0.72067420 9.6012957 0,02039035 NS NS
ENSMUSG00000053317 Sec61b 0.72271861 8.6464277 0,02394618 NS NS
ENSMUSG00000032115 Hyou1 0.72542206 111.90740 0,000261459 0,01184542 0,009999353
ENSMUSG00000029992 Gfpt1 0.73010342 128.74029 0,000181333 0,01169538 0,009300651
ENSMUSG00000025823 Pdia4 0.73578803 99.75082 0,001245015 0,01889195 0,01937855
ENSMUSG00000027274 Mkks 0.73859129 10.1872651 0,01692847 NS NS
ENSMUSG00000024807 Syvn1 0.74925435 12.5888520 0,01491 NS NS
ENSMUSG00000022136 Dnajc3 0.75897388 107.92095 0,001613378 0,02941396 0,0224176
ENSMUSG00000036752 Tubb4b 0.76208405 128.46661 0,00137721 0,02961917 0,02992061
ENSMUSG00000021270 Hsp90aa1 0.76412886 487.61517 3,71512E-09 0,01869743 0,01748928
ENSMUSG00000020571 Pdia6 0.76530663 115.60622 0,001585131 0,03259344 0,02569314
ENSMUSG00000022403 St13 0.76694814 183.61440 0,000159801 0,02485634 0,01006615
ENSMUSG00000020048 Hsp90b1 0.77268789 333.65080 3,62403E-06 0,02545554 0,01035048
ENSMUSG00000038991 Txndc5 0.78533220 21.6381701 0,01223921 NS NS
ENSMUSG00000057177 Gsk3a 0.79325964 9.3428609 0,04547464 NS NS
ENSMUSG00000020964 Sel1l 0.80117569 39.8227204 0,01028297 NS NS
ENSMUSG00000003814 Calr 0.80683350 280.52363 5,73728E-05 NS 0,03557301
ENSMUSG00000014905 Dnajb9 0.81077498 13.6103914 0,04756119 NS NS
ENSMUSG00000027248 Pdia3 0.82023420 266.61918 0,000844391 NS 0,03250441
ENSMUSG00000025980 Hspd1 0.83155570 39.3474128 0,005265961 NS NS
ENSMUSG00000027006 Dnajc10 1.20432485 36.3353195 0,004916254 NS NS
ENSMUSG00000030102 Itpr1 1.23316873 37.0042251 0,003676613 NS NS
ENSMUSG00000057329 Bcl2 1.24012846 12.5516511 0,03456831 NS NS
ENSMUSG00000041417 Pik3r1 1.30733252 390.20651 7,967E-08 0,0122928 0,02680347
ENSMUSG00000033538 Casp4 1.32151410 14.8300797 0,01902025 NS NS
ENSMUSG00000093904 Tomm20 1.34528949 74.42460 0,001110166 0,01932788 0,02751819
ENSMUSG00000020303 Stc2 1.52415772 5.5675880 0,02826457 NS NS
ENSMUSG00000036052 Dnajb5 1.99140600 4.632706 0,03248627 0,04135138 NS

≥1,500
≥1,200
Fold change ≤0,833
≤0,666
≤0,500
 Table S2 – Antibodies used for flow cytometry staining and cell sorting

Antibody Conjugated Clone Dilution Manufacturer

CD45.2 PB 104 1/200 Sony Biotech
CD45.1 PE A20 1/100 BD Biosciences
TCRb APCCy7 H57-597 1/200 Sony Biotech
CD4 PECy7 RM4-5 1/200 Sony Biotech
CD8 PECy5 53-6.7 1/200 BD Biosciences
CD44 APC IM7 1/200 Sony Biotech
CD62L FITC MEL-14 1/200 Sony Biotech
CD19 PECy7 1D3 1/200 BD Biosciences
IgM APC RMM-1 1/200 Sony Biotech
CD11b APCCy7 M1/70 1/200 Sony Biotech
CD11b AF488 M1/70 1/200 Sony Biotech
CD115 PECy7 AF598 1/200 eBiosciences
Ly6C FITC AL-21 1/200 BD Biosciences
Ly6G PerCPCy5.5 1A9 1/200 Sony Biotech
Sca-1 PE D7 1/200 Sony Biotech
CD117 APC ACK2 1/200 eBiosciences
CD117 AF700 ACK2 1/200 eBiosciences
CD150 PECy5 TC15-12F12.2 1/200 Biolegend
CD48 APCCy7 HM48-1 1/200 Sony Biotech
CD34 FITC RAM34 1/200 BD Biosciences
CD127 APCeF780 A7R34 1/200 eBiosciences
TER-119 PE TER-119 1/200 BD Biosciences
CD71 FITC C2 1/200 BD Biosciences
CD16/32 PECy7 93 1/200 Sony Biotech
LIN (CD3, PB 17A2; RB6-8C5; 1/10 (1/20 for Biolegend
Ly6G/Ly6C; M1/70; RA3-6B2; cultured cells)
CD11b; Ter-119
B220; TER-
1119)
CD3e APC 145-2C11 1/200 BD Biosciences
CD11b APC HL3 1/200 BD Biosciences
CD19 APC 6D5 1/200 Sony Biotech
CD19 APCCy7 1D3 1/200 BD Biosciences
 Figure S1

HSC MPP HPC-2 HPC-1

6 2.0 6 40
ctrl ctrl ** ctrl
ctrl
fsn fsn *** fsn *
fsn

Nb of cells (x104)
Nb of cells (x104)

Nb of cells (x104)
Nb of cells (x104)
1.5 30
4 ** 4

1.0 20

2 2
0.5 10 ****
*
0 0.0 0 0
3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks

CMP GMP MEP CLP

150 30 150 25
ctrl ctrl ctrl ctrl
fsn fsn * fsn fsn
Nb of cells (x105)

** 20 *

Nb of cells (x105)
Nb of cells (x105)

Nb of cells (x105)
***
100 20 100
15

*** **
10
50 * 10 50
5

0 0 0 0
3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks
Figure S2
A B
LSK MPP HPC-2 HPC-1
ctrl
60 15 30 80 **** fsn

Frequency in LSK cells

Frequency in LSK cells

Frequency in LSK cells

Cells in femur (x103)

60
40 10 20

40

20 5 10
20

0 0 0 0
3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks
 Figure S3

A
RBC Hemoglobin Hematocrit
10 *** *** * 20 ** *** *** 70
*** **** ** ctrl
Number of cells (x106/μL)

60 fsn

Concentration (g/dl)
8
15

Frequency (%)
50
6 40
10
4 30

20
5
2
10

0 0 0
3 wks 6 wks 12 wks 3 wks 6 wks 12 wks 3 wks 6 wks 12 wks

B C
Erythroid differenciation
Bone marrow Spleen 3,55 0,26
Number of erythroblasts (x106)
Number of erythroblasts (x106)

4 250
ctrl ctrl
fsn 200 fsn 4,78
ctrl
3

150 89,7
2
49,1 1,56
100
*
1
50 * fsn 31,1

0 0 CD71
ProE Baso Poly Ortho ProE Baso Poly Ortho 15,2
FSC
Figure S4

A HSC HPC-2 HPC-1

25 100 80 ctrl
fsn
20 80

% of BrdU+ cells
60
% of BrdU+ cells

% of BrdU+ cells
15 60
40
10 40
20
5 20

0 0 0
3 12 3 12 3 12
Age (weeks) Age (weeks) Age (weeks)

B C
HSC
100 Ctrlctrl Ctrlfsn
Ctrlctrl 10,9 12,5
80 Ctrlfsn
4,6 5,8
Cells (%)

60

40
80,0 80,8
Ki67
20
Hoechst 33342
0
G0 G1 S/G2/M
Figure S5

A B
Survival 6th round 7th round
100 100 100 Ctrlctrl
Ctrlctrl
Ctrlfsn Ctrlfsn
80 80 80
% of live mice

Survival (%)

Survival (%)
60 60 60

40 40 40

20 20 20

0 0 0
1st 2nd 3rd 4th 5th 6th 0 10 20 30 40 50 0 5 10 15
Time (days) Time (days)

C
1st round 2nd round 3rd round

Re
Re

Re
la
la

la
tiv
tiv

tiv
e
e

e
co
co

co
n
n

n
tri
tri

tri
bu
bu

bu
lls

lls

lls
tio
tio

tio
ce

ce

ce
n
n

n
d

to

d
to

to
loi

loi

loi
T
T
ye

ye

T
ye
ce
ce

ce
m

m
lls
lls

lls
to

to

to
ion

ion

ion
ut

ut

ut
rib

rib

rib
nt

nt

nt
co

co

co
ive

ive

ive
lat

lat

lat
Re

Re

Re
Relative contribution to B cells Relative contribution to B cells Relative contribution to B cells

4th round 6th round 7th round

Re
Re

Re
lat
lat

lat
ive
ive

ive
co
co

co
nt
nt

nt
rib
rib

rib
lls

lls

lls
ut
ut

ut
ce

ce

ce
ion
ion

ion
d

d
to
loi

loi

loi
to

to
T
ye

ye

ye
T

T
ce
m

m
ce

ce
lls
lls
to

to

to

lls
ion

ion

ion
ut

ut

ut
rib

rib

rib
nt

nt

nt
co

co

co
ive

ive

ive
lat

lat

lat
Re

Re

Re

Relative contribution to B cells Relative contribution to B cells Relative contribution to B cells

Ctrlctrl Ctrlfsn
Figure S6

2500
Normalized expression (AU)

2000
300

200

100

0
Sca-1 cKit CD150 CD48 Ttc7a
Figure S7

A B
MPP HPC-2 HPC-1
3.0 1.5
1.5 ctrl
Proliferation index (AU)

Proliferation index (AU)

Proliferation index (AU)
2.5 ctrl fsn
2.0 1.0 1.0 fsn
1.5
0.5 0.5
1.0

0.5
0.0 0.0
0.0
Aggresome
None 0,6 1,2 None 0,6 1,2 None 0,6 1,2
TM (μg/mL) TM (μg/mL) TM (μg/mL)

C Atf6 Ire1α Perk eIf2α Xbp1 Chop Atf4

100 250 40 200 300 30 150

Normalized expression (AU)

Normalized expression (AU)

Normalized expression (AU)

Normalized expression (AU)
Normalized expression (AU)

Normalized expression (AU)

Normalized expression (AU)

80 200
30 150
200 20 100
60 150
20 100
40 100
100 10 50
10 50
20 50

0 0 0 0 0 0 0
ctrl fsn ctrl fsn ctrl fsn ctrl fsn ctrl fsn ctrl fsn ctrl fsn

D E
HSC HPC-1 HPC-2
60
DMSO
* TM 0,6
Annexin V+ cells (%)

*
40
* TM 1,2 ctrl fsn
Hsp70
20 *
Gapdh
0
ctrl fsn ctrl fsn ctrl fsn

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