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\dateposted{2023-12-21}
\datepublished{2024-03-29}
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%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{A tribute to Fran\c{c}ois Gros, a founding father of molecular biology}
%%\def\cdr@specialtitle@french{Un hommage \`a Fran\c{c}ois Gros, un des p\`eres de la biologie mol\'eculaire}
%\makeatother

\title{Messenger \mbox{RNA} in differentiating muscle  cells---my experience
in Fran\c{c}ois Gros' lab in the 1970s and 80s}

\alttitle{Les \mbox{ARN} messagers des cellules du  muscle pendant leur diff\'erenciation --- mon exp\'erience comme membre du laboratoire de Fran{\c{c}}ois Gros dans les ann\'ees 1970s et 80s}

\author{\firstname{Margaret} \lastname{Buckingham}\CDRorcid{0009-0007-7290-9239}}
\address{Department of
Developmental and Stem Cell Biology,  CNRS UMR 3738,  Institut Pasteur,
25 rue du Dr Roux, 75015 Paris, France}
\email{margaret.buckingham@pasteur.fr}

\keywords{\kwd{Fran\c{c}ois Gros}
\kwd{\mbox{RNA}}
\kwd{Myogenesis}}

\altkeywords{\kwd{Fran\c{c}ois Gros}
\kwd{ARN}
\kwd{Myogen\`{e}se}}

\def\thanksname{Note}
\thanks{This article follows a symposium held on 25 April 2023 at the
Institut Pasteur in tribute to Fran\c{c}ois Gros.}

\begin{abstract} 
I joined Fran\c{c}ois Gros' laboratory as a postdoc at the end of 1971
and continued working with him as a research scientist until 1987, when
I became an independent group leader at the Institut Pasteur. In the
early 1970s, it was the beginning of research in his lab on muscle cell
differentiation, as a model eukaryotic system for studying \mbox{mRNAs} and
gene regulation. In this article, I recount our work on myogenesis and
mention the other research themes in his lab and the people concerned.
I remained in close contact with Fran\c{c}ois and pay tribute to him as
a major figure in French science and as my personal mentor who provided
me with constant support.
\end{abstract}

\begin{altabstract} 
J'ai rejoint le laboratoire de Fran\c{c}ois Gros en tant que
postdoctorante \`{a} la fin de l'ann\'{e}e 1971 et j'ai continu\'{e}
\`{a} travailler avec lui en tant que chercheuse jusqu'en 1987, date
\`{a} laquelle je suis devenue chef de groupe ind\'{e}pendante \`{a}
l'Institut Pasteur. Au d\'{e}but des ann\'{e}es 1970, son laboratoire a
commenc\'{e} \`{a} \'{e}tudier la diff\'{e}renciation des cellules
musculaires, comme un syst\`{e}me mod\`{e}le eucaryote permettant
d'\'{e}tudier les ARNm et la r\'{e}gulation des g\`{e}nes. Dans cet
article, je retrace nos travaux sur la myogen\`{e}se et mentionne les
autres th\`{e}mes de recherche de son laboratoire ainsi que les
personnes concern\'{e}es. Je suis rest\'{e}e en contact \'{e}troit avec
Fran\c{c}ois et je lui rends hommage en tant que figure majeure de la
science fran\c{c}aise et en tant que mentor qui m'a apport\'{e} un
soutien constant.
\end{altabstract}

\maketitle

{\vspace*{5pt}}

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\section{My arrival in France}

I joined the laboratory of Fran\c{c}ois Gros as a postdoc in October,
1971. Having just completed a D.~Phil. (Ph.D.) on histone modifications
with M. Ord in the Biochemistry department of Oxford University, I
wanted to work on messenger \mbox{RNA} (\mbox{mRNA}), the discovery of which had
inspired us as students. I heard Fran\c{c}ois, who had played an
important role in this discovery, speak at a FEBS meeting in Montreux
and with some trepidation had asked him if there would be a possibility
of working in his lab. With his habitual kindness, he encouraged me to
come. Alas, subsequently, there was no reply from Fran\c{c}ois to my
letter(s). Finally after a phone call, Genevi\`{e}ve \mbox{Antolini,} who was
already his efficient assistant, activated a formal letter of
acceptance and, with a \mbox{postdoctoral} fellowship from the Royal Society,
I came to Paris with my husband Richard who joined the lab of \mbox{Marianne}
Grunberg-Manago at the Institut de \mbox{Biologie} Physico-Chimique (IBPC).

Fran\c{c}ois had left the IBPC in 1968 and had his laboratory in the
science faculty of the University of Paris where he was professor of
the new discipline of molecular biology. The lab was located in a wing
of the huge new concrete complex at Jussieu which housed the faculty.
The site was not a very welcoming environment and Fran\c{c}ois' lab at
that time was also not a very convivial place for a newly arrived
postdoc. It was difficult to find out who was doing what in the absence
of joint group meetings. Fran\c{c}ois proposed that I work with Daniel
Caput, a Ph.D. student. The Ph.D. cursus involved a th\`{e}se de
troisi\`{e}me cycle and then a th\`{e}se d'\'{E}tat, which could last
for many years, unlike the three-year Ph.D. that I had just \mbox{completed}
in the UK. Fran\c{c}ois Gros, like others who had
\mbox{characterised} \mbox{mRNA} in
bacterial systems, wanted to explore \mbox{mRNA} function and regulation in
eukaryotes. Through his close contacts with the Weizmann \mbox{Institute,} he
knew of David Yaff\'{e}'s work on muscle cells. Myoblasts can be
separated from large multi{\hbreak}nucleated muscle fibres and will proliferate
in culture at low density. When the culture becomes confluent, they
will fuse together, accumulate muscle proteins and form fibres, thus
providing a model system for the \textit{in vitro} study of cell
differentiation. When I arrived, Fran\c{c}ois had recently decided to
adopt this model. Daniel had begun work on muscle primary cultures and
Denise Luzzati, in a separate effort, was studying rat L6 and L8 muscle
cell lines isolated by D.~Yaff\'{e}.

\section{Fran\c{c}ois Gros' lab in the Institut Pasteur}
In 1972, Fran\c{c}ois moved most of his lab to the Institut Pasteur. In
retrospect, this move cast a shadow over the lab during my first year
as a postdoc, because of uncertainty about who would move with him.
Jacques Monod had just been appointed Director of the Institute and had
decided, with admirable realism, that the effort required to
re-establish the failing financial situation was incompatible with
directing his own research. He therefore proposed to Fran\c{c}ois to
return as Director of the Laboratory of Biochemistry, located on the
6th floor of the new Molecular Biology building, now
called the Monod building. This floor had been planned for Monod's
laboratory and included a large office, with elegant light wood
fittings. Fran\c{c}ois Jacob, and other previous members (J.-P.
Changeux, H. Buc, M. Schwartz, etc.) of the Monod team had laboratories
located on other floors, constituting the molecular biology department
of the Institut Pasteur. It was a stimulating scientific environment
with exciting internal and external seminars. In Fran\c{c}ois' lab, we
now had memorable weekly meetings when the different groups would
present their work. This ranged from transcriptional regulation of
phage lambda (P. Kourilsky, finishing his th\`{e}se d'\'{E}tat), of the
SV40 virus (M. Yaniv, just returned from a postdoc with Paul Berg) to
\mbox{mRNA} translation (J. Thibault, J.-C. Lelong, D. Lazar), and to muscle
cell differentiation worked on initially in the small group constituted
by D. Caput and \mbox{myself.} Fran\c{c}ois presided and would ask questions
and construct 
possible theories, leading to lively discussion. His
scientific
insights were valuable and his
encouragement precious. These
were convivial occasions with coffee and croissants from the best
patisserie in the nearby rue de Vaugirard. I remember that
Fran\c{c}ois, who spoke and wrote elegantly, did not like anglicisms
even in scientific French and would construct a phrase to avoid such
pollution, for example a whole sentence for ``Southern blot'', a
technique developed by Ed. Southern for characterising DNA.
Fran\c{c}ois' mastery of English included erudite words; he explained
that as a student he had decided it was essential to know English and
therefore memorised several pages of the dictionary every day. Among
his many attributes he had a remarkable memory! A few years later M.
Yaniv became director of his own research unit in the building and P.
Kourilsky left for a postdoc with B. Mach in Switzerland. After
Fran\c{c}ois Gros' election to the chair of cellular biochemistry at
the Coll\`{e}ge de France in 1973, he established a second laboratory
there with a focus on neural differentiation. Some of his
collaborators, including those working on \mbox{mRNA} translation in Pasteur,
moved to the Coll\`{e}ge to help establish this new laboratory. M.
Cr\'{e}pin formed a research group in the Pasteur lab, working on the
initiation of \mbox{mRNA} transcription, with an interest in the MMTV virus.
M. Jacquet joined Fran\c{c}ois' laboratory in Pasteur, with a group
working on the function of chromatin in the transcriptional regulation
of eukaryotic cells, thus complementing the interest of J. Yaniv in the
bacterial chromosomal protein HU. D. Caput left the lab when he had
finished his thesis and joined the pharmaceutical company of Sanofi. 

\section{The expansion of research on the muscle model of cell
differentiation}
Gradually Fran\c{c}ois recruited more researchers working on muscle
differentiation, notably Bob Whalen, a postdoc who had worked with Paul
Doty in Harvard. Bob and I shared a small office with another American
postdoc, John Merlie, who worked with
\mbox{J.-P.~Changeux,} in collaboration
with Fran\c{c}ois, on the synthesis of the acetylcholine receptor in
muscle cell cultures. We had fun discussing everything from science to
politics. The office was opposite Fran\c{c}ois' secretariat and it was
impressive how many people, from the most eminent to the most humble,
came to seek his advice. Bob Whalen carried out important 
research on 
muscle protein isoforms, notably actins 
and myosin heavy and light
chains. He, like me, was able to form a research group in Fran\c{c}ois'
lab, with postdocs such as Gill Butler-Browne and Shin-ichi Takeda, as
well as Lev Ovchinikov from A. Spirin's institute in the then Soviet
Union. We both benefitted from very good people who had applied to work
with Fran\c{c}ois, in my case Sataro Goto who had worked on muscle cell
hybrids with Nils Ringertz, and then Adrian Minty. Woodring (Woody)
Wright was another remarkable American postdoc who came to work with
Fran\c{c}ois, developing his original idea of using heterokaryons,
rather than hybrid cells, to look at the dominance of the muscle
phenotype on other cell backgrounds. Woody was a fervent collector of
``junk'' from the pavements and flea markets of Paris which accumulated
in his small lab space, viewed by Fran\c{c}ois with amused tolerance.
Another muscle researcher, recruited later by Fran\c{c}ois, was Marc
Fiszman who had developed a model system for regulating muscle cell
behaviour, using thermosensitive retroviral infection in chick cell
cultures. Domenico Libri, Didier Montarras and Vincent Mouly carried
out research with him on muscle cell differentiation and on the muscle
isoforms of\break tropomyosin.

\section{Professional women in research}
From the beginning of my time in Pasteur, thanks to Fran\c{c}ois, I
benefitted from the collaboration of Arlette Cohen who worked with me
for 30 years, first as a technician and latterly as a research
engineer. In addition to valuable practical help at the bench, she also
was my mentor for Parisian  fashion---disapproving of my Scottish
clothes---, and has remained a close friend. She is Fran\c{c}ois'
second cousin. His wife, Dani\`{e}le, also worked as a technician in
the lab, first with P. Kourilsky, then with M. Cr\'{e}pin and later
with M. Jacquet and then Marc Fiszman. She said, laughingly, that this
was the only way to see more of Fran\c{c}ois and to have some contact
with his professional life. He did indeed work non-stop. For him,
French holidays provided a peaceful time to concentrate on 
writing---reports and also books, mainly about the major advances of
molecular biology and also his own experiences in the \mbox{development} of
this new discipline. I remember an occasion when the lift stopped on
the  6th floor and three small boys rushed out down the corridor,
escaping their child minder, with cries of ``maman''. They did not go
the other way 
towards Fran\c{c}ois' office, having been taught that
their father
should not be disturbed.
This said, although Fran\c{c}ois
did not spend much time playing with them, he was clearly a benevolent
father. In Parisian bourgeois society, that a daughter should work as a
technician or as a scientist was totally acceptable. Educated women
were encouraged to pursue their career as well as having a family. This
was in contrast to other countries at that time where this was frowned
upon. I remember when my first son was born in 1980, Fran\c{c}ois sent
me congratulations and a large bunch of\break flowers!

\section{Early experiments on \mbox{mRNAs} during muscle cell differentiation}
My research as a postdoc, initiated with Daniel Caput, involved pulse
labelling \mbox{RNA} as muscle cells differentiated. Our experiments at that
time were rather reminiscent of those carried out by Fran\c{c}ois using
\textit{E.~coli} in Jim Watson's lab in Harvard~\cite{1}, when he was
sent there as a biochemist to explore unstable \mbox{RNA} (\mbox{mRNA}) by Jacques
Monod. Daniel had decided to make use of a source of muscle from foetal
calves, available from the Vaugirard slaughter house, not far from
Pasteur, which still existed in the first half of the 1970s. The
advantage of course was the quantity of the material even if the stage
of development was not precisely controlled. Many eukaryotic \mbox{mRNAs}
contain poly(A) at the three prime end, and we could isolate them from
bulk ribosomal \mbox{RNA} using poly(U)-sepharose 
columns. We also fractionated
cytoplasmic extracts on sucrose gradients to separate the heavier
polysomal fraction from the so-called ribonucleoprotein (RNP) fraction
nearer the top of the gradient. These kinds of experiments led us to
describe increased \mbox{mRNA} stability in cultures where proliferation was
lower and cell fusion to form myo{\hbreak}tubes had been initiated. With Sataro
Goto we explored a possible role of the length of poly(A) tails on \mbox{mRNA}
stability. We also noted that certain \mbox{mRNAs} could be chased from the
RNP compartment into polysomes~\cite{2}. An echo of these results came
38 years later when we showed that adult muscle satellite cells
sequester the transcript of the myogenic determination  gene
\textit{myf5} in RNP particles prior to their activation and
differentiation when the \mbox{mRNA} is translated~\cite{3}. At the time we
did not have a way of identifying individual \mbox{mRNAs}. A major step
forward came with the availability of  systems---wheat germ or
reticulocyte lysates---for \textit{in vitro} translation of \mbox{mRNA}
populations,
followed by identification of radioactively labelled
protein products. Separation of denatured proteins by two-dimensional
gel electrophoresis, made it possible to distinguish different isoforms
of the contractile proteins and thus plot the appearance of \mbox{mRNAs}
encoding muscle isoforms as cells differentiated~\cite{4}. This work
was carried out with Philippe Daubas, my first Ph.D. student, who later
returned to my lab with a CNRS position, after postdoctoral experience
elsewhere. By the end of the 1970s, the synthesis of complementary DNA
(cDNA) sequences, obtained by reverse transcription of \mbox{mRNA} populations,
made it possible to perform experiments of differential hybridisation.
Nabeel Affara, a postdoc in Fran\c{c}ois' lab, working with a
teratocarcinoma derived myoblast cell line isolated by members of
Fran\c{c}ois Jacob's lab, documented changes in \mbox{mRNA} complexity with a
newly transcribed group of \mbox{mRNAs} now present in polysomes, as these
cells formed muscle fibres~\cite{5}.

\section{The advent of specific cDNA probes for\newline single 
\mbox{mRNAs}---myogenesis \textit{in vivo}}
After the development of DNA reverse transcription and its application
to \mbox{RNA}, a major technological breakthrough came with the cloning of
cDNAs. When introduced into bacteria in a plasmid vector,
characterisation of the bacterial colonies permitted the isolation of
single cDNA sequences which could then be used as molecular probes for
specific \mbox{mRNA} sequences. Such DNA cloning of potentially pathological
sequences was a subject of concern and in 1974 a moratorium was
introduced by the scientific community. This was lifted in 1975 after
an important conference at Asilomar in California had laid down
security guidelines for such experimentation~\cite{6}. In my group, we
began to clone cDNAs of \mbox{mRNAs} for muscle actins and myosins. This was
not easy, partly because the technology was new and partly because we
had to follow strict security. The experiments took place in a P2
facility in the second basement (not accessible with the usual lift)
and all the equipment and solutions used had to be autoclaved before
being taken back to the lab. Experiments had to receive prior approval
from a scientific committee set up by the Ministry of Research. We
decided to use mouse skeletal muscle as a source of \mbox{mRNA}. Cloned 
probes
would make it possible to look at skeletal muscle  formation \textit{in
vivo} as
well as in
cultured cells and the mouse is a suitable animal
model. We adopted the mouse C2 muscle cell line, isolated by David
Yaff\'{e}, as  our \textit{in vitro} mammalian system. The first muscle
\mbox{mRNA} to be cloned, by Adrian Minty, was a muscle $\rmalpha$-actin
sequence~\cite{7}. The similarities between actin isoforms meant that
the coding sequences cross-hybridised, but fortunately the 3$'$
non-coding sequences of these and other \mbox{mRNAs} are distinct. With cloned
cDNA probes, we could now look, by hybridisation to Northern blots, at
specific \mbox{mRNAs} during C2 cell differentiation. This approach, backed up
by two-dimensional gel analysis of the products  of \textit{in vitro}
\mbox{mRNA} translation, gave precise information about the presence of
contractile protein \mbox{mRNAs}, showing, for example, the accumulation of
the \mbox{mRNA} for the embryonic myosin isoform, MLC1emb, at the onset of
cell fusion, before that of the fast isoforms MLC1F and 
MLC3F~\cite{8}. \textit{In vivo}, in fetal mouse skeletal muscle, two
$\rmalpha$-actin type sequences co-accumulate, coding the cardiac
isoform of the adult heart as well as the predominant isoform of adult
skeletal muscle~\cite{9}. In the C2 muscle cell line, cardiac actin
\mbox{mRNA} is also expressed as soon as the cells begin to differentiate.
This co-expression of actin genes is also seen in developing heart
muscle. Expression of the same isoforms in the two types of striated
muscle applies also to myosins, for example, the so-called MLC1emb
isoform of developing skeletal muscle is also present in the atria of
the adult heart (MLC1A)~\cite{10}. Instead of co-expression, sequential
expression of developmental and adult isoforms is also a strategy, as
documented for the myosin heavy chain \mbox{mRNAs} by Andr\'{e} Weydert in my
group~\cite{11}. Interestingly this is reflected in the organisation of
the corresponding genes. Using interspecific mouse lines for genetic
mapping, in collaboration with J.-L. Guenet in Pasteur, we could show
that actin and myosin light chain genes are dispersed in the
genome~\cite{12}, while sequentially expressed myosin heavy chain genes
are present in the same\break locus~\cite{13}.

\section{Characterisation of muscle genes}
In addition to characterising modes of expression, cDNA probes also
made it possible to isolate genes and examine their structure. Thus
Benoit Robert, who had joined my group as a Ph.D. (th\`{e}se
d'\'{E}tat) student, after working with M. Jacquet, and had 
cloned the
first myosin cDNAs, isolated the locus that encompasses exons 
encoding
the fast myosin MLC1 and MLC3 proteins. He showed how these are
generated by the use of distinct promoters with differential
splicing~\cite{14}, thus providing one of the first examples of this
phenomenon which leads to the generation of isoforms of many
contractile proteins. On my first visit to Japan, when I spoke in place
of P. Kourilsky at an international conference, I had learned that
Yo-ichi Nabeshima was working on the same gene in chicken. I visited
his rather obscure lab in Nigata and, after the obligatory polite
exchanges with his professor, we had an exciting time comparing results
which showed the same gene structure. We agreed to try to synchronise
publication of our papers which were accepted by \textit{Cell} and 
\textit{Nature}. Y.~Nabeshima subsequently discovered the myogenic 
regulatory factor,
Myogenin, and became a leading Japanese scientist, with a professorship
in Kyoto University. My second Ph.D. student, Serge Alonso and a
British postdoc, Ian Garner, continued the work on actin coding
sequences and their evolution~\cite{15}. As a result of a genetic
analysis of actin genes, a mutation in the cardiac actin locus  of
\textit{BALB/c} mice led to new insights into an upstream regulatory
region of the cardiac actin gene~\cite{16}. Later, we isolated distinct
skeletal and cardiac muscle enhancer sequences located at the
5$'$-end of the gene~\cite{17}. In the 1980s, initial studies on the
transcriptional regulation of muscle genes depended on transcriptional
``run on'' experiments in the C2 cell line and then transfection of
differentiating cultured cells with reporter plasmids controlled by
candidate regulatory sequences.  \textit{In vivo} experiments with
transgenes only came later. In addition to Ian, two other able British
postdocs joined my group in the 1980s, Roger Cox who did ``run on''
experiments~\cite{18} and also worked on MHC genes and Paul Barton, who
was briefly a permanent Inserm researcher, working on myosin light
chain genes, notably coding for\break MLC1emb~\cite{19}.
\looseness=1

\section{Transcription factors that activate\newline myogenesis}
Little by little we obtained clues about the regulation of myogenesis.
Characterisation of the muscle cell  \textit{in vitro} system and of
the onset of muscle gene expression when cells begin to differentiate
was a necessary first step. However, the ultimate aim was to 
understand
how this is regulated, how a differentiated tissue phenotype 
is
acquired during
development. One approach was to identify sequences
that control muscle gene expression and systematically progress
upstream from there. Another was to identify potential candidate
upstream regulators by a shot in the dark approach. During the
formation of skeletal muscle in the fruit fly  \textit{Drosophila}, a
muscle specific homeobox factor had been described as an upstream
regulator of myogenesis. With Benoit Robert, we therefore decided to
look for the mammalian homologue and cloned a sequence for what we
called Hox7~\cite{20}. This, and its homologue Hox8, are now known as
Msx1 and Msx2. Msx1 is expressed at a low level in some myogenic
progenitors in the mouse embryo, however these factors are mainly
important in patterning the limb mesenchyme and at other non-myogenic
sites during development. Benoit subsequently explored their functions
in his own lab. A major breakthrough came in 1987 when Hal Weintraub
and colleagues identified a cDNA sequence present in muscle cells,
which, when transfected into fibroblasts, would convert them to
myogenesis. They described the bHLH transcription factor encoded by
this sequence as myogenic determination factor MyoD1~\cite{21}. This
approach would not have worked for most tissue types where more than
one ``pioneer'' transcription factor is required for tissue
differentiation. In the case of skeletal muscle, other factors are also
implicated in the activation of muscle genes, however MyoD plays an
over-riding role. We now know that there are four members of the MyoD
family. They are specific to skeletal muscle cells and all have the
capacity, when overexpressed, to force many cell types into myogenesis.
Myogenin and Mrf4, as well as MyoD, are implicated in muscle cell
differentiation, whereas MyoD and Myf5 (with Mrf4 in the early embryo)
expressed in myoblasts act as myogenic determination factors before
muscle tissue forms. Towards the end of the 1980s, David Sassoon, an
American postdoc, joined my lab and \mbox{established} the newly developed
technique  of \textit{in situ} hybridisation on tissue sections to
analyse gene expression during embryonic development. At that time, we
had a bet with Hal Weintraub who was convinced that MyoD would be
expressed first at the onset of myogenesis in the mouse embryo. David
showed that this was not the case and that the gene for another member
of the family, Myogenin, was expressed earlier~\cite{22}. Using this
technique, David and Gary Lyons, another American postdoc in my group,
mapped out the temporal-spatial expression of muscle genes during
cardiac and skeletal muscle development (ex.~\cite{23}). At that time
this was a new technical approach, and we organised an EMBO course  on
\textit{in situ} hybridisation in 1990.

\section{Subsequent research in my lab}
In my own laboratory, we subsequently showed that Myf5 is expressed
prior to the first formation of skeletal muscle~\cite{24} and much of
our subsequent work focussed on the function and regulation of the gene
for this myogenic determination factor (ex.~\cite{25}). We also studied
the role of Pax3 (ex.~\cite{26}), a transcription factor which is not
specific to myogenic progenitor cells, but which orchestrates the entry
of cells into the myogenic programme. It controls cell fate choices
in the mesodermal structure of the somite, myogenic progenitor cell
proliferation and migration from the somites to the limbs, and directly
activates sequences regulating the transcription of  \textit{myf5}.
Most of our research on the regulatory hierarchy that controls the
onset of myogenesis concerned the embryo, but we also, with Didier
Montarras, worked with muscle stem cells in the adult, so called
satellite cells which are responsible for muscle regeneration~\cite{27}
and which are governed by a similar genetic hierarchy. As a result of
our interest in the expression of myosin genes, initiated in
Fran\c{c}ois' lab, and due to an insertion site effect on a myosin
transgene, we discovered the second heart field~\cite{28}. This is an important
source of cardiac progenitors which we showed constituted a second
myocardial cell lineage~\cite{29}. Cardiogenesis, as well as
myogenesis, became a major theme of my lab. From the 1980s, as a result
of the tools of molecular biology and later of molecular genetics also,
it became possible to examine gene expression, regulation and function
during the development of the organism. It was an exciting time. More
recently the centre of interest has shifted to stem cell biology, with
less emphasis on the developmental aspect which underlies stem cell
behaviour and more on adult tissue regeneration, with the potential for
therapeutic applications.

\section{External recognition and scientific\newline meetings with
Fran\c{c}ois} Thanks to Fran\c{c}ois' support, I had obtained a
permanent position in the CNRS in 1975. There was less competition then
in the small community of molecular biologists and the recommendation
of a ``grand patron'' counted a lot. I had acquired an international
reputation and
had also been successful in gaining grants, notably at that time
financial support from the American Muscular Dystrophy Association
(MDA), which benefitted his  lab. Recognition of my scientific
contribution also owed a lot to Fran\c{c}ois. He introduced young
scientists from his lab to the French community and would on occasion
drive us to meetings in Paris. To be a passenger in Fran\c{c}ois' car
was not without risk. I remember an occasion when he was driving round
the Etoile and had entered the inside lane nearest the Arc de Triomphe.
He was so concentrated on a scientific discussion that he did not move
towards an outside lane to exit and we continued going round and round
until an alarmed passenger reminded him of our destination! From 1982,
he did not co-sign many of my publications and before that he
encouraged me to present my work at international meetings, such as the
big FEBS meeting in Paris in 1975. I remember that David Yaff\'{e},
also speaking in the session on cell differentiation, noticed my
nervousness and offered me a piece of chocolate, his remedy for stress
before speaking. David played a major role in the development of the
field of myogenesis, organising EMBO workshops that continue today. He
organised a first EMBO workshop on the subject at the Shoresh kibbutz
in Israel in 1975 and then again in 1980. I attended both meetings with
Fran\c{c}ois. At that time, we were a small number of researchers in
the world working on muscle cell differentiation. Clashes of opinion at
the meetings were frequent between the Americans Irv Konigsberg and
Howard Holtzer, both strong personalities, who, together with David
Yaff\'{e}, were the great men of muscle cell culture, primary cultures
from chick in the case of the former and mouse cell lines for David.
Molecular regulation, which interested Fran\c{c}ois, was in its
infancy. His quiet interventions at the meeting had a calming effect on
the cell biologists! In general, Fran\c{c}ois was always courteous and
very rarely expressed anger. After the 1980 meeting in Shoresh, I had
planned to stay in Jerusalem to explore the old city. I remember
Fran\c{c}ois' concern at leaving  me---expecting my first child in a
few months' time---,
in a rather down at heel hotel run by Palestinians
beside the Jaffa gate. Later, in  1988, we organised an EMBO meeting on
the island of Bendor near Bandol in the South of France. By that time
the community working on myogenesis was bigger and the focus of
interest was muscle gene regulation. During my time in Fran\c{c}ois'
lab, other memorable meetings that we attended were organised by his
colleague and friend Marianne Grunberg-Manago in the then Soviet Union,
as part of Russian-French scientific collaboration, and also on the
Greek island of Spetsai where regular summer schools were held with
leading international  figures giving lectures on molecular biology. I
remember Francis Crick sitting at the back of the lecture theatre in
the school asking lethal questions in a piping voice ``But my dear boy,
what about{\ldots}''. The Spetsai summer schools continue
today.

In 1987, I was made laboratory head in Pasteur with an independent
research Unit. I had decided it was time to move on and was thinking of
applying for my own group in CNRS centres in the Paris region. When I
discussed this with Fran\c{c}ois he was reluctant that I leave.
Fran\c{c}ois Jacob had just decided to give part of his space on the 
4th floor to his ex-pupil Jean-Fran\c{c}ois Nicolas and advised that I
be allocated half of the  6th floor. Fran\c{c}ois generously decided to
follow this advice. The Scientific Council of Pasteur agreed and I thus
became officially independent while benefitting from the continuing
proximity of my  6th floor colleagues.

\section{Fran\c{c}ois Gros' responsibilities in French science, in
addition to those as lab director}
In 1975, Jacques Monod became fatally ill and asked Fran\c{c}ois to
take over as director of the Institut Pasteur. He occupied the
director's offices on the other side of the Pasteur campus and was less
present in the lab. However, he continued to attend lab \mbox{meetings} and
would come to the  6th floor at the end of the day when we could
discuss science with him. During this period, he wrote an important
report with Fran\c{c}ois Jacob and Pierre Royer, commissioned by the
French president V. Giscard d'Estaing, on the impact that the new
discoveries of molecular biology were likely to have on the community.
After the election of Fran\c{c}ois Mitterrand as president,
Fran\c{c}ois was appointed scientific advisor to the prime ministers,
Pierre Mauroy and then Laurent Fabius, a post that he occupied from
1981 to 1985. During this period, he played an important political role
in the organisation of scientific research in France 
and recognition of 
the scientific and medical implications of molecular biology and
emergent biotechnologies. In addition to his work for the government,
in the context of muscle research, Fran\c{c}ois raised awareness of the
importance of this with the directors of the French AFM (Association
Fran\c{c}aise contre les Myopathies) and became the first president of
their scientific council from 1986. The AFM continues to provide
precious financial support to research labs in France today. Despite
all the demands on his time during the 1980s, Fran\c{c}ois still came
to the lab regularly, while the group leaders ensured its functioning
and maintained the scientific life that Fran\c{c}ois had
instituted.

The end of the 1980s was a difficult time for Fran\c{c}ois because of
the propagation of the AIDS virus and the disastrous contamination of
blood banks. The government was blamed for not taking action sooner and
Fran\c{c}ois, as scientific advisor, was also accused in the subsequent
legal proceedings. Many scientific colleagues around the world wrote
letters in his support and indeed little was known about the virus at
the time. In the end, Fran\c{c}ois was cleared of any mis-doing. In
1991, he was elected Secretary of the French Acad\'{e}mie des Sciences
of which he had been a member since 1979. Together with the President,
two ``Secretaries'', one for biological sciences and one for physical
sciences, run the Academy. It is an important position and his election
demonstrated the esteem in which he was held by his fellow scientists
in all disciplines. He now had his main office in the Academy and
occupied the position until 2000, continuing to be active in the
Academy until the end of his life. He continued as head of his lab in
Pasteur and also as professor at the Coll\`{e}ge de France, until he
reached retirement age (70) in 1995. He still came to the lab on the
6th floor of the Monod building and enjoyed hearing about the
experiments that were ongoing. He also always remained very supportive
of those who had worked with him. I greatly benefitted from his
thoughtful interest in my research, his kindness and the valuable
advice and practical assistance that he generously gave. For example,
my election to the Acad\'{e}mie des Sciences in 2005 owed much to his
discrete support. At the end of his life, he still maintained his
interest in research and indeed, even after the Covid-19 epidemic had
meant that he could no longer go to the Academy and communication was
limited to phone calls, he wanted to hear about new scientific results.

\section*{Declaration of interests}
The author does not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and has
declared no affiliations other than her research institution.

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