THE MAMMALIAN DIAPHRAGM AS AN EVOLUTIONARY NOVELTY

1. The Mammalian Diaphragm
as an Evolutionary Novelty
Tatsuya Hirasawa
Department of Earth and Planetary Science
Graduate School of Science
The University of Tokyo, Tokyo, Japan
Co-evolution of Life and Environment during the Key Geological Transitions

2. How Did Evolutionary Novelties Arise?
Developmental mechanism
- cell migration
- gene regulatory network
- molecular crosstalk between cells
Evolutionary novelty = new homology
(= new developmental constraint maintaining the morphological unit)
Fossil record
- morphological transition
- variability in the past
- parallel gain/loss
(directionality in evolutionary changes)
Placoderm pectoral spine
from the Lower Devonian of Japan
Shark embryo
(Scyliorhinus torazame, stage 25)

3. Evolutionary Novelty: Diaphragm
Introduction
The diaphragm
- Skeletal muscle settled deep in the thorax
- Innervated by the phrenic nerve from the
neck level
- Important function in breathing
- Conserved in all the mammals
- No comparable structure in non-
mammalian vertebrates

4. Adaptation for Higher Oxygen Uptake
Introduction
Modified from Duncker (1978)
Powered by
the diaphragm
Birds Mammals
Different solutions to the trade-off between surface area and compliance

5. Aim of This Study
Introduction
How did this evolutionary novelty arise?
→ Study from the perspective of developmental biology

6. Pleuroperitoneal fold (PPF)
Medial outgrowth of
lateral body wall
Embryonic Development of the Diaphragm
Introduction
Migratory muscle precursor
(MMP) cells
Migrating somitic cells
Developmental origin: combination of PPF and MMP

7. Pleuroperitoneal fold (PPF)
Medial outgrowth of
lateral body wall
Migratory muscle precursor
(MMP) cells
Migrating somitic cells

8. Lateral Body Wall (LBW)
Body Wall Development
Hirasawa, Fujimoto, Kuratani (2016)
ph: phrenic nerve
ppf: pleuroperitoneal fold
trs: transverse septum
ht: heart
dc: ductus cuvieri
Caudad shift of the heart
Mouse embryo
E11.5
E10.5

9. Body Wall Development
Question
In mammals, the lateral body wall is transformed during development,
to bring the cell of the cervical level to the level of the boundary
between the heart and the liver
Is this mammalian-specific?
(or shared in other amniotes?)

10. Development of the Neck in the Chicken
Body Wall Development
HH stage 11 HH stage 19
HH stage 22 HH stage 30
Ventral view

11. Visualizing Body Wall Development
Body Wall Development
Method: chicken-quail chimera
HH stage 11–12
Somatopleure at a cervical somite level
was exchanged homotopically with
somatopleure of a quail
QCPN (quail cell marker)
immunostaining
16 hours
after grafting

12. Development of the Neck in the Chicken
Body Wall Development
Hirasawa, Fujimoto, Kuratani (2016)
Transplantation of
cervical LBW
at 8th somite level
(mid-neck level)

13. Development of the Neck in the Chicken
Body Wall Development
Hirasawa, Fujimoto, Kuratani (2016)
Cervical lateral body wall later
extends into the thorax,
concomitant with the caudal
transposition of the heart

14. Infolding of Cervical Lateral Body Wall
Body Wall Development
Chicken
Mouse
← Chicken
Infolding of the cervical lateral body wall
into the thorax
← Mouse
Infolding of the diaphragm primordium
into the thorax
- Infolding of lateral body wall is shared
- Diaphragm had evolved based on this
shared developmental process

15. Pleuroperitoneal fold (PPF)
Medial outgrowth of
lateral body wall
Migratory muscle precursor
(MMP) cells
Migrating somitic cells

16. Developmental Process of Muscles
Muscle Development
Migratory Muscle Precursor (MMP) (e.g., Dietrich, 1999)
- Dissolving the segmentation pattern of somites
- Migrating a long distance from ventral part of the
dermomyotome
- Specific gene expressions (Hgf and c-Met signaling
pathway, Pax3, Lbx1)
MMP-derived muscles
Pax3 expression in chicken

17. Question
- MMP cells migrate a long distance with specific gene expressions
- The hypobranchial muscles, diaphragm and limb muscles develop
from MMP, unlike other skeletal muscles
What is the ancestral condition of MMP distribution?
Muscle Development

18. Nerves of the MMP-derived Muscles
Position of the nerve can be a proxy for the position of the MMP cell origins
Comparative data from various species
Muscle Development

19. Brachial Plexus & Skeletal Morphology
- Basic organization of brachial plexus
is shared
- Its position correlates with
the cervico-thoracic transition of ribs
Paleontology

20. Brachial Plexus in Fossil Species
Positions of brachial plexus can be reconstructed
in fossil species, based on rib morphology
Paleontology

21. Brachial Plexus Shifted Caudally
Number of cervical vertebrae: 5 à 7
Position of brachial plexus had been
shifted caudally by two somites
Paleontology

22. Partial Duplication of the Brachial Plexus
Evolutionary origin of the phrenic nerve
- C4–C5 in mammals = vestiges of ancestral C4–C5
- Phrenic nerve = subscapular nerve of the ancestor
Paleontology

23. Brachial Plexus Patterns
Paleontology
Hirasawa & Kuratani (2013)
Cervico-thoracic boundary

24. Hirasawa & Kuratani (2013) Hypothesis
Paleontology
The diaphragm likely evolved from a shoulder muscle,
through a partial duplication of the primordial cell population

25. Developmental Constraint?
Paleontology
- Diaphragm-MMP cells are required to migrate into PPF
- Diaphragm-MMP and forelimb-MMP develop at the shared developmental field
→ Position of forelimb cannot be changed (= number of cervical vertebrae )
Massetognathus (MCZ 3691)
Dimetrodon (MCZ 1365)
5 cervical vertebrae 7 cervical vertebrae

26. Hoxc5
Mouse Chicken
MMP Duplication
Subscapular Muscle as ‘Proto-diaphragm’
Paleontology

27. 245 Million Years Ago
Paleontology
First appearance of the diaphragm
(Brink, 1956; Hillenius & Ruben, 2004; Buchhiltz et al., 2013)
Regionalization of the trunk

28. Drop of atmospheric oxygen level enhanced the canalization of diaphragm development
→ developmental constraint on the number of cervical vertebrae as seven
Atmospheric Change
Paleontology

29. Summary
- The diaphragm becomes settled deep in the thorax, through the
infolding of the cervical lateral body wall, which is also seen in
other amniotes
(Hirasawa, Fujimoto & Kuratani, 2016: Development, Growth & Differentiation)
- The diaphragm likely evolved from a shoulder muscle, through a
partial duplication of the primordial cell population
(Hirasawa & Kuratani, 2013: Journal of Anatomy)

30. References
Website
http://www.s.u-tokyo.ac.jp/en/people/hirasawa_tatsuya/
Selected papers (researches on the diaphragm)
Hirasawa, T., and S. Kuratani. 2013. A new scenario of the evolutionary
derivation of the mammalian diaphragm from shoulder muscles.
Journal of Anatomy 222: 504–517.
Hirasawa, T., S. Fujimoto, and S. Kuratani. 2016. Expansion of the neck
reconstituted the shoulder-diaphragm in amniote evolution.
Development, Growth & Differentiation 58: 143–153.
Hirasawa, T., and S. Kuratani. 2018. Evolution of the muscular system in
tetrapod limbs. Zoological Letters 4: 27.