Lecture 7

MODULE: Muscle development and regeneration

MODULE: Flatworm regeneration

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 Myoblasts are cells that are
Myoblasts à skeletal muscles in culture committed to becoming muscle
but have yet to show signs of
Myocytes differentiation.
(post-mitotic)

• (A) MyoD and Myf5 are required for lineage specification of muscle cells
• (C) Myoblast recognition and alignment mediated by glycoproteins and cadherins
• (D) During cell fusion, Myogenin (a MRF required for muscle cell differentiation) becomes active and turns on muscle-
specific genes.
Dev Biol 12 e, Further Development 17.5: Maturation of2 Muscle
Myotome à Skeletal muscle

1. Commitment of myotome cells to myoblasts. Cells express bHLH TFs- Myf5 and MyoD

2. Terminal differentiation of committed skeletal myoblasts into myotubes. Cells express

bHLH TFs- Myogenin and MRF4

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 Myogenic genes are only expressed in primordia that give rise to muscle tissue

Expression of MRFs

MRF4 mRNA
in somites
Mouse embryo

Kassar-Duchossoy et al., Nature, 2004

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 MRFs = bHLH (basic helix-loop-helix) Transcription Factors

MyoD protein structure

NH2 transactivation basic HLH transactivation COOH

DNA binding dimerization

MyoD:E2A heterodimer

E-box = enhancer
6 bp
CANNTG
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 Functional analysis of MRFs
MRF: Myogenic regulatory factors
MRFs: MyoD, Myf5, MRF4, Myogenin

MyoD expression
Fibroblast Muscle cell
(Cell culture Transdifferentiation
=> in vitro) (reprogramming)

Ectopic expression à each myogenic factor is sufficient

to drive muscle development in vitro

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 Functional analysis of MRFs
Loss-of-function assay: mouse mutants (= in vivo)

1. Myf5-/- ; MyoD-/- (double mutants): lack myoblasts, skeletal muscle does not form
à Myf5 and MyoD control the determination of myoblast fate
(Note: single mutants of Myf5 or MyoD have normal skeletal muscle)

2. Myogenin-/- : normal numbers of myoblasts but muscle does not form

à Myogenin (and MRF4, data not shown) controls muscle differentiation

Conclusion: MRFs are required for muscle development

determination differentiation
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https://doi.org/10.1242/dev.124.13.2507 DOI: 10.1016/0092-8674(93)90621-v
 Expression of myogenic factors during muscle development

embryo

adult

somite
Myoblasts
cells
Satellite cells
= muscle stem
cells
Myotubes/
Myofibers
Myf5
MyoD
Myogenin
Mrf4
temporal order of expression of myogenic factors

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Partial figure from: https://cshperspectives.cshlp.org/content/4/2/a008342.full.pdf+html
Ectopic expression of Myogenin– premature expression
How could we achieve
premature expression
of Myogenin?
Myf5 gene Myogenin gene Myf5En Myogenin

'early' 'late' 'early'

enhancer enhancer enhancer

Knock-in

Myogenin-/- + Myogenin knock-in ( + Myf5-/-)

à Normal numbers of myoblasts
endogenous Myogenin gene Myf5En Myogenin
But no muscle differentiation
(no rescue)
'late' 'early'
enhancer enhancer

à Myogenin promotes muscle differentiation

only when expressed at the correct time DOI: 10.1242/dev.124.13.2507
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 Expression of Myogenin in differentiated muscles causes muscle atrophy
Extra info

chromatin regulator

transcriptional co-factor
Myogenin Myogenin
expression expression
repressed activated
ubiquitin-ligases
target proteins
to the proteasome
for degradation

=> Myogenin expression

has to be restricted to a
specific time window
during muscle development
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Moresi et al., Cell. 2010 Oct 1;143(1):35-45.
Spatial and Temporal control of the expression of developmental genes
is essential for normal development. One of the ways by which gene
expression is spatially and temporally controlled is through gene-
specific enhancers.

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Muscle regeneration and repair

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 Skeletal muscle growth is negatively regulated by myostatin
Muscle growth:
• increase in the number of muscle fibers
• Increase in the size of muscle of fibers – due to newly generated myocytes
fuse with existing muscle fibers

Myostatin: made by developing & adult muscle

“Herculean phenotype”
Myostatin
(TGFß factor)
T
• number of myofibers
(more: hyperplasia)
• size of myofibers
(more: hypertrophy)

myostatin mutant Further Development 17.5:

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Maturation of Muscle
 Satellite cells function as Muscle stem cells
quiescent
satellite cell Injury, Exercise dividing
satellite cell

Satellite cell (muscle stem cell; actively dividing

reside between the basal lamina satellite cell
and the plasma membrane of muscle fiber)

daughter stem cell differentiating

replaces mother daughter cell
stem cell forms new
muscle cells 14
Gilbert 11th ed. - Fig.17.35
 Asymmetric cell division

Muscle fiber

satellite cell daughter

differentiating
= mother stem cell asymmetric stem cell
daughter
cell division

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Exercising mouse
Polarized activation ->
muscle fiber = stem
Asymmetric cell division
cell niche

FGF2 FGF2 (Fibroblast growth factor)

FGFR1 (FGF receptor)
FGF signaling
pathway
satellite
cell

mitosis

muscle repair & growth

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 Signaling by p38α/β MAPK is involved in the exit of satellite cells from quiescence, asymmetric division of
satellite cells, specification of myoblasts (by inducing expression of Myf5/MyoD) and differentiation of satellite
cells in vivo.
Pax-7
Muscle
Fiber
(stem cell) Secretes
FGF-2
Myf5/MyoD
myoblast
Polarized activation
è Asymmetric cell
division
Transient amplification
of muscle precursors
by cell division

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Bentzinger & Rudniki, 2014. https://www.nature.com/articles/nm.3499
 Increased p38 signaling impairs self-renewal of aged satellite cells
Pax-7
Muscle
Fiber
(stem cell) Secretes
FGF-2

Polarized activation
è Asymmetric cell
division
Transient amplification
of muscle precursors
by cell division

• loss of self-renewed satellite cells further reduces the regenerative capacity of the aged muscle tissue
• decreased proliferation and differentiation potential of aged muscle precursors 18
Review Question

What is the correct flow of skeletal muscle development in a vertebrate

embryo?

a. myotome à myoblast à myotube à muscle fiber

b. myoblast à myotome à myotube à muscle fiber
c. myotome à myoblast à muscle fiber à myotube
d. myotome à myotube à myoblast à muscle fiber

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What happens if you introduce the gene myoD into fibroblast cells growing in culture?

a) The fibroblasts will express muscle-specific proteins, fuse, and form functional muscle in
culture.

b) Nothing will happen because fibroblasts are derived from a different cell population in
the embryo than are the muscle precursors that respond to myoD.

c) The fibroblasts will undergo apoptosis, due to the abnormal effects of myoD on their
normal differentiation.

d) myoD will activate expression of myogenin, but the muscle-specific genes will not be able
to activate.

e) Nothing will happen because fibroblasts do not have the genes required to become
muscle.
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 Module: Flatworm Regeneration

Suggested Reading:
Developmental Biology 12th edition:
Chapter 22: Regeneration, Defining the problem of regeneration p 643-645
Stem cell mediated regeneration in flatworms p659-662

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 Regeneration

“Regeneration is the reactivation of development in post-embryonic life

to restore missing and damaged tissues.”
Scott Gilbert

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What mechanisms drive Regeneration?

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 Flatworm

Hydra ~0.5 cm long

Hercules and Hydra

525 BC
Sci Signal. 2010 Jun 22;3(127):pe21. doi:
10.1126/scisignal.3127pe21.Wnt signaling in For every head chopped off, Hydra
axial patterning and regeneration: lessons regenerated a new one
from planaria.De Robertis EM.
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FYI: https://www.perseus.tufts.edu/Herakles/hydra.html
 Modes of tissue regeneration in mammals

Stem-cell mediated regeneration: Compensatory

Skin, muscles, gut, blood cells regeneration: Liver

differentiated cells Stem cells cells remain differentiated

self-renewal
differentiation
Gilbert 11th ed. - Fig.22.2

Gilbert 11th ed. - Fig.22.2 25

To study regeneration, you need a good model organism

What would be the desired features of the experimental organism?

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To understand mechanisms that drive regeneration, you need a good model organism

Features of a desired experimental system:

• Regenerates robustly
• Regenerates quickly
• Can be grown in lab
• Can study cells
• Can study genes

Planarian flatworm: classic regeneration model

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Planarian flatworm: members of the phylum
Platyhelminthes (Platy, flat; helminth, worm)
Complex anatomy- brain,
eyes, musculature, intestine,
epidermis

Schmidtea mediterranea- model organism

• Bilaterally symmetrical
• Distinct antero-posterior polarity
• Distinct dorso-ventral polarity

Planaria ~ 40 different cell types-

Neurons, epidermal cells, muscles, intestinal cells etc.
Ventral view of the gut (green), nerve chords (red) coming from
the head 28
 𝑆. 𝑚𝑒𝑑𝑖𝑡𝑒𝑟𝑟𝑎𝑛𝑒𝑎 reproduce asexually

http://www.ub.edu/planaria/index.html
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𝐏𝐥𝐚𝐧𝐚𝐫𝐢𝐚𝐧 𝐅𝐥𝐚𝐭𝐰𝐨𝐫𝐦

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31
Anterior (Head)

Posterior (Tail)

Thomas Hunt Morgan (1866-1945)

Nobel prize for Medicine or Physiology (1933)

Dalyell wrote in 1814 that planarians

appear to be “immortal under the edge of the32knife”.
1

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2

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In the regeneration lectures, will discuss:

1. What is the cellular basis of regeneration in planarian flatworms?

2. What is the molecular basis of regeneration polarity in planarian

flatworms?

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 Flatworm- head regeneration

Amputation Wound healing Blastema formation Cell differentiation

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Cell production during planarian regeneration is accomplished by a pluripotent stem cell population of neoblasts

Neoblasts are
Neoblasts (Neoblasts):
• distributed throughout the
Pluripotent somatic stem cells in flatworms
body
that produce all the new tissues during
• excluded from the head
planarian regeneration
and the pharynx

• 20%-30% cells are neoblasts

• Only mitotic cells in planarians
• After injury, migrate to the wound site
Dapi (blue) labels all cell nuclei; and generate post-mitotic descendants to
Neoblasts (red)labelled with an RNA form the regenerative blastema- epidermal progenitors,
probe to soxP-2 – located everywhere gut progenitors, muscle progenitors, neural progenitors
posterior to the eye spots and excluded
from pharynx
etc.

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 Are neoblasts required for regeneration?

=> worm loses

regeneration ability
(immediately)

=> degenerates
(within 2-7 weeks)
loss of neoblasts

-> neoblasts are required to regenerate new tissues after amputation

-> neoblasts are required for homeostasis (normal tissue renewal- planarian tissues undergo
constant turnover)

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Cell production during planarian regeneration is accomplished by a pluripotent stem cell population of neoblasts

Do neoblasts contain
lineage-restricted cells that are
collectively pluripotent or are there
stem-cells that are also pluripotent at
a single-cell level?

S. mediterranea the worm has also

Injection of a single (clonogenic) regained its
neoblast from a non-radiated
donor worm into an irradiated host
regeneration ability
worm ~1% success rate 41
Clonogenic neoblasts (cNeoblasts): neoblasts that produce colony of post-mitotic cells that can differentiate
into neuronal, muscle, intestinal and all other cell types required for homeostasis and replacing missing
tissues during regeneration.

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 irradiated
donor
host

clonogenic
neoblast

Do the regenerated worms

have the genotype of the host or the donor worm?

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 Formation of different cell types during regeneration

Blastema cells differentiate into

different cell types

Specialized neoblasts (fate-specified

cells)=> undifferentiated blastema cells
(postmitotic = no cell division)

wound

cNeoblasts (pluripotent stem cells )

(undergo cell division)
drawing adapted after:
Reddien PW.
Development. 2013,140:951-7.
Specialized progenitors 44
and regeneration.
Different classes of neoblasts with different developmental potential
cNeoblast
Extra info

piwi-1+ cell marker + neoblast is

committed to differentiation

As stem cells exit the cell cycle (post-mitotic)

, their commitment to a lineage is likely to occur.

Some progenitor cells for specific tissues and

lineages have been found and others are not yet
associated with a given lineage (no connecting lines).

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https://doi.org/10.1038/s42003-022-03905-9
 Neoblasts produce the cells of the blastema

Apoptotic cells
at amputation
site (purple)

cNeoblasts
migrate
to wound site
(blue)

Division progeny
of neoblasts
form blastema
(red, green;
start expressing
genes specific to
Fate-specified Luca Gentile et al. Dis. Model. Mech. 2011;4:12-19
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neoblasts) © 2011. Published by The Company of Biologists Ltd
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 Neoblasts migrate and repopulate regions that lost neoblasts
Neoblasts
express Neuroblasts
Neuroblasts smedwi-1 are restricted
die upon to regions
exposure to Neoblast progeny protected from
X-rays - Not seen after X-rays
4dpi in irradiated?
nuclear marker
(Hoechst)
Dpi= 4 days post irradiation
Migration of neoblasts dpa = days after
and progeny after head amputation
head amputation

Migrating neoblast
progeny display shielded FISH to look
extensive cellular region at Nb markers
protrusions

doi: 10.1242/dev.154971 47
 Migration of neoblasts restores regenerative ability
Tissue death Regeneration

(dpi = days after irradiation)

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doi: 10.1242/dev.154971
SUMMARY:

• In planarian flatworms, regeneration occurs by forming a regenerative blastema

that consists of undifferentiated cells produced from clonogenic neoblasts
(cNeoblasts). These cells then differentiate to form the missing tissues.

• cNeoblasts are also required for normal tissue renewal.

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 Wild type

Wild type Wild type

Some cool cuts to try in the next lab

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Peter Reddien