Lecture 3: Musculoskeletal Development

Slide 1: Title slide: Musculoskeletal Development

Main points: Covers the Derivatives of Mesodermal Germ Layer section of Chapter 6,
Chapter 9 (Skeletal System), and Chapter 10 (Muscular System)

Slide 2: Cross section showing the different subdivisions of the mesodermal germ layer and a
listing of the origins of the different types of muscle
Main points:
1. It is important to note that some smooth muscle (for the pupil and the mammary and
sweat glands) is derived from ectoderm and not mesoderm, demonstrating that there
is an exception to every rule.

Slide 3: Illustration of a 4-week embryo showing external features

Main points:
1. Somites form in the paraxial mesoderm from the occipital to the sacral regions.
2. In the head region, paraxial mesoderm is organized into somitomeres. These
structures represent less well-defined somites, but they still represent segmentation of
paraxial mesoderm, and they contribute to skeletal muscle of the head and neck.

Slide 4: Scanning electron micrograph of a chick embryo showing somite formation along the
neural tube
Main points:
1. Somites form from unsegmented paraxial mesoderm that has a fibroblast-like
appearance.
2. Unsegmented mesoderm condenses, becomes epithelial rather than fibroblastic, and
forms a new segment called a somite.
3. Regulation of the process is controlled by a segmentation clock that maintains the
correct periodicity.

Slide 5: Cross section through a somite (somites appear as pairs; one on each side of the
neural tube)
Main points:
1. Somites are somewhat donut-shaped with a small lumen.
2. At this stage, somite cells appear epithelial rather than fibroblastic.

Slide 6: Scanning electron micrograph showing a cross section through a pair of somites
Main points:
Note the epithelial nature of somite cells.

Slide 7: Cross sections showing somite differentiation

Main points:
1. Somites differentiate into muscle, bone, and dermis of the skin.

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 2. The ventral region forms the sclerotome that contributes to formation of the
vertebrae. Some sclerotome cells also migrate into the parietal layer of lateral plate
mesoderm to contribute to rib formation.
3. Ventrolateral and dorsomedial regions form muscle precursor cells. Some of the
ventrolateral cells migrate into the parietal layer of lateral plate mesoderm to
contribute limb and body wall muscles. Both of these regions have cells that migrate
ventral to the dorsal portion of each somite where they form back muscles.
4. The dorsal region of the somite contributes cells that form the dermis of the back.
5. Note that, for each of these regions, the cells lose their epithelial appearance and,
once again, become fibroblast-like.

Slide 8: Illustration showing molecular signals that regulate somite development

Main points:
1. Somite differentiation is regulated by a multitude of signals emanating from
surrounding structures.
2. An important gene product to stress is sonic hedgehog (SHH) secreted by the
notochord and floor plate of the neural tube. SHH is important because it plays an
integral role in so many processes, as in this example.

Slide 9: Illustrations showing the external appearance of a 4-week embryo and HOX genes
that pattern the body axis in fruit flies (Drosophila) and humans
Main points:
1. This slide reminds students that somites differentiate into different structures in
different regions. This is most obvious by noting that vertebrae from the cervical
region are quite different morphologically from those in thoracic and lumbar regions,
etc.
2. Patterning for the somites and their differentiation is regulated by HOX genes, which
are conserved from fruit flies.
3. Fruit flies have one set of these genes that regulate head, thorax, abdomen, etc.,
development, whereas humans have four copies of these genes located on four
different chromosomes that do the same for us.
4. Note that HOX gene expression is in part regulated by retinoic acid (RA). Alterations
in retinoid levels produced by Accutane (for treatment of cystic acne) and etretinate
(topical retinoid for acne) are teratogenic and cause a variety of birth defects. The
mechanism for induction of some of these defects may involve abnormal expression
of HOX genes.
Interesting point: Fruit flies do not have well-developed brains and cerebral hemispheres;
therefore, HOX genes are expressed only so far as the hindbrain region in humans. Other
homeobox genes (not of the HOX class) regulate most of our brain development.

Slide 10: Cross sections showing differentiation of muscle cell precursors in the somite.
Main points:
1. Two muscle-forming regions develop: a ventrolateral unit that contributes cells to
both the parietal layer of lateral plate mesoderm and the dermomyotome (area ventral

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 to dermis-forming cells) and a dorsomedial unit that only contributes cells to the
dermomyotome.
2. Cells migrating from the ventrolateral unit cross the lateral somitic frontier to enter
the lateral plate mesoderm.
3. The lateral somitic frontier represents a border between cells that remain in the axial
region and form the primaxial domain and cells that migrate across the border to enter
the abaxial domain.
4. Note that the ventrolateral unit of muscle cell precursors contributes cells to both
domains.
5. Some sclerotome cells also migrate across the lateral somitic frontier.
6. Muscle cells in the abaxial domain form limb and body wall muscles and receive
many of their signals for differentiation from lateral plate mesoderm, whereas cells in
the primaxial domain form back muscles and receive their differentiation signals from
the neural tube and notochord.
7. Sclerotome cells that migrate into the abaxial domain form the cartilaginous portion
of the ribs; those in the primaxial domain form the bony portion of each rib and the
vertebrae.
8. The lateral somitic frontier also represents a dividing line between dermis formed for
the back from the dermatome and dermis formed from lateral plate mesoderm for the
body wall.
Interesting note: The above description of muscle cell development and origin differs from the
old idea of epaxial (from epimeres) and hypaxial (from hypomeres) origins. The major difference
is that ventrolateral muscle cells contribute to both epaxial (limb and body wall muscles) and
hypaxial (back muscles) muscles. The terms epaxial and hypaxial are still accurate for describing
the location of muscles, but not for muscle origins. Thus, epaxial (back) muscles are still
innervated by dorsal primary rami, while hypaxial (limb and body wall) muscles are innervated
by ventral primary rami.

Slide 11: Illustrations showing the segmental origin of skeletal muscles and their innervation
Main points:
1. Head musculature develops from 7 somitomeres, which are segmented whorls of
paraxial mesoderm.
2. Musculature of the axial skeleton, limbs, and body wall is formed from somites.
3. Some of these muscle cells migrate some distance to arrive at their definitive
locations (e.g., limb muscle cells).
4. Skeletal muscles of the axis, body wall, and limbs are innervated by spinal nerves
from the segments of origin of the muscles. These nerves will follow the muscles
wherever they may migrate.
5. A spinal nerve forms at the intervertebral foramen by the union of dorsal (sensory)
and ventral (motor) roots. Each nerve then divides almost immediately into a dorsal
and ventral primary ramus, each of which carries motor and sensory fibers.
6. Epaxial muscles (back muscles) are innervated by dorsal primary rami; hypaxial
muscles (limb and body wall muscles) are innervated by ventral primary rami.

Slide 12: Illustrations showing segmental innervation of dermatomes

Main points:

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 1. Just as with muscles, dermatomes are innervated by spinal nerves associated with
their origin.

Slide 13: A child with Poland sequence (absence of the pectoralis minor and part of the major,
usually the sternal head)
Main points:
1. Absence of a muscle is relatively common, for example, the palmaris longus or
serratus anterior, but seldom debilitating.
2. In Poland sequence, absence of the pectoral muscles may include absence or
displacement of the nipple, which can be problematic for females.

Slide 14: A child with prune belly syndrome (absence of abdominal musculature)
Main points:
1. Most investigators attribute this loss of musculature to accumulation of fluid in the
abdominal cavity due to obstructive urinary problems. Abdominal distension is said
to thin the muscles, causing necrosis.

Slide 15: Cross section of somite differentiation to focus on sclerotome differentiation

Main points:
1. Sclerotome cells form from the ventral portion of each somite.
2. Those cells remaining in the primaxial domain form vertebrae and the bony portion
of each rib.
3. Those migrating across the lateral somitic frontier form the cartilaginous portions of
the ribs.

Slide 16: Illustrations showing vertebrae formation

Main points:
1. Sclerotome cells from each pair of somites migrate around the neural tube.
2. Each vertebra is formed from pairs of adjacent somites; thus, the caudal half of one
joins with the cranial half of another. This process is called resegmentation.
3. Resegmentation reorients the relationship between vertebrae and their myotomes so
that muscles derived from the myotomes now bridge adjacent vertebrae, allowing the
muscles to move the corresponding intervertebral joint.
4. Intervertebral discs have a fibrous ring (annulus fibrosis) formed from sclerotome
cells and a cushiony center (nucleus pulposis) derived from notochord cells.

Slide 17: Illustrations of a somite and a vertebra

Main points:
1. It is amazing that from the dispersed sclerotome a unique vertebra is formed with its
tremendously complex shape.
2. HOX genes regulate this amazing example of patterning.

Slide 18: Components of the skull

Main points:

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 1. As indicated.

Slide 19: Origin of neural crest cells: spinal cord regions

Main points:
1. Neural crest cells form from neurectoderm cells at the crests of the neural folds.
2. Neural crest cells in spinal cord regions migrate from the folds after neural tube
closure.
3. Spinal cord crest cells migrate and differentiate into many cell types, including
melanocytes, ganglia for the gut tube, dorsal root ganglia, etc.

Slide 20: Origin of cranial neural crest cells

Main points:
1. Cranial neural crests also form at the crests of the neural folds but leave the folds
before they close.
2. These cells migrate into the head and neck area to form bones of the face and skull
and to the heart to participate in septation of the outflow tract.

Slide 21: Origins of bones of the head and neck

Main points:
1. Flat bones (membranous neurocranium) of the skull are derived from paraxial
mesoderm (red) and neural crest (blue).
2. All bones of the face (viscerocranium) are derived from neural crest.
3. Laryngeal cartilages are derived from lateral plate mesoderm (yellow).

Slide 22: Origins of bones forming the cartilaginous neurocranium (chondrocranium, base of
the skull)
Main points:
1. Neural crest cells (blue) form the base of the skull cranial to the pituitary fossa.
2. The pituitary fossa also represents the cranialmost extension of the notochord.
Cranial to this point lies prechordal plate mesoderm; posterior to this point lies the
notochord. Hence the nomenclature: prechordal and chordal regions of the
chondrocranium.
3. Paraxial mesoderm (red) forms the base of the skull posterior to the pituitary fossa.

Slide 23: Membranous bone formation

Main points:
1. Flat bones of the skull form directly from fibroblasts by deposition of bony spicules.
2. This mechanism is different than the base of the skull (chondrocranium) that forms a
cartilage model of the bone first followed by ossification (endochondral ossification).

Slide 24: Cranial sutures

Main points:

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 1. Sutures of fibrous tissue form between bones of the skull to allow for expansion as
the brain grows. They also allow the bones to overlap each other (molding) as the
babys head passes through the birth canal.
2. Spaces where two or more sutures intersect are called fontanelles. The anterior one
forms the soft spot on a baby's head.
3. In most cases, the anterior fontanelle closes by 18 months, while the posterior one
closes by 1-2 months. Some stay open into adulthood.

Slide 25: Definition and characteristics of craniosynostosis (premature closure of one or more
cranial sutures)

Slides 26 & 27: Examples of skull defects resulting from craniosynostosis

Main points:
1. Scaphocephaly: Sagittal suture.
2. Brachycephaly: Coronal sutures.
3. Plagiocephaly: Unilateral coronal and lambdoid sutures.
4. Cloverleaf skull: Most cranial sutures.

Slide 28: Examples of syndromes caused by mutations in fibroblast growth factor receptors
(FGFRs)
Main points:
1. FGFs and their receptors are involved in bone formation.
2. Mutations in different FGFRs cause different syndromes and may involve the head,
face, and/or the long bones.
3. Mutations in the same receptor can cause different syndromes depending on where
the mutation occurs.

Slide 29: Child and adult with achondroplasia caused by mutations in FGFR3
Main points:
1. Mutations in these receptors can affect the head, the face, and the long bones.

Slide 30: Illustration of early limb bud formation

Main points:
1. Forelimb bud appears first at 4 weeks as a proliferation of lateral plate mesoderm
and overlying ectoderm.
2. Hindlimbs appear approximately 1 day later.
3. HOX genes specify location of the limbs along the body axis.
4. TBX5 specifies forelimb; TBX4 specifies the hindlimb.
5. TBX genes are called T-Box genes and are transcription factors.

Slide 31: Embryos summarizing the external appearance of the limbs during development
Main points:
1. The forelimb develops ahead of the hindlimb by about 24 hours. By 8 weeks, the
limbs are well formed with fingers and toes.

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
Slide 32: Scanning electron micrograph of a developing limb bud showing the apical
ectodermal ridge (AER)
Main points:
1. Once proliferation of the lateral plate mesoderm creates initial limb outgrowth,
ectoderm at the tip (apices) of the limb becomes columnar and forms a ridge, the
AER.
2. The ridge is an important signaling center and controls limb growth in a
proximodistal (away from the body) direction.
Interesting point: Limbs represent one of the ultimate examples of the complexities of
patterning. Not only are there differences in fore- and hindlimb patterns, but each limb has its
own patterning along the proximodistal, anterior-posterior, and dorsoventral axes. Knowledge
about factors that regulate these processes has been facilitated by limbs from laboratory animals,
such as chicks and mice, which can be manipulated in vivo and grown in vitro. Thus, limb
development has been a favorite topic of embryologists for decades, and what has been learned
about the embryology and genetic regulation of limb development has greatly assisted our
understanding of other developmental processes.

Slide 33: Illustration showing regulation proximodistal limb growth

Main points:
1. FGFs secreted by the AER create the progress zone in the immediately adjacent
mesoderm.
2. FGFs keep cells in the progress zone proliferating and prevent their differentiation.
3. As the limb grows, cells at the back of the progress zone are moved farther away
from the influence of FGFs secreted by the AER.
4. As FGF concentrations decrease around these cells, they begin to form condensing
mesenchyme, the first step in differentiating into cartilage.

Slide 34: Mechanism for regulating anterior-posterior limb patterning

Main points:
1. A group of cells in the posterior border of the limb near the AER called the zone of
polarizing activity (ZPA) regulates patterning of the limb in the anterior-posterior
direction (preaxial to postaxial: thumb to little finger).
2. The master gene for this regulation is sonic hedgehog (SHH), whose product is
secreted by these cells.
3. Secretion of SHH creates a gradient with the highest concentrations on the little
finger side and lowest on the thumb side.
4. The persons hand exhibits polydactyly and also almost mirror-image duplication.
5. Mirror-image duplication is the result of experiments where a region of ZPA is
grafted from the posterior region of one limb to the anterior region of a normal limb
in chick embryos. The experiment creates two ZPAs in the same limb and a mirror-
image duplication of a chick's digits.
Interesting point: These chick experiments were conducted in the 1950s when investigators
discovered the AER and ZPA and knew they produced factors regulating these phenomena; they
just could not identify the factors with the techniques they had at the time.

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
Slide 35: Limbs showing development of cartilages over time
Main points:
1. Bones of the limbs form by endochondral ossification; they are laid down as
cartilage models that then ossify.
2. The first sign of cartilage differentiation is condensation of limb mesenchyme.
3. Bones develop in a proximodistal direction, such that the pubic bones develop first,
then the femur, etc. (Note: As the limb grows and cells are left behind, they become
located farther and farther away from the influence of the AER and can begin to
differentiate. That is why differentiation starts proximally.)

Slide 36: Illustrations showing endochondral bone formation

Main points:
1. Cells condense and then differentiate into chondrocytes that establish a cartilage
model of the bone.
2. Osteoblasts then replace the cartilage cells and ossification occurs.
3. Growth plates remain for a time to produce cartilage cells and continued bone
growth.
4. Most growth plates close (stop cell production) after the growth spurt during puberty.

Slide 37: Illustrations showing digit formation

Main points:
1. Programmed cell death (apoptosis) in 4 areas of the AER creates 5 digits.
2. Digits continue growing under the influence of the remaining parts of the AER that
cover each one.
3. Interdigital tissue is removed by programmed cell death to create 5 separate digits.
Interesting point: Programmed cell death or apoptosis occurs in many developing tissues. There
is a separate cascade of genes regulating this phenomenon, and disruption of this process results
in birth defects (e.g., syndactyly in the digits). Intuitively, cell death appears counterproductive to
tissue growth and development, but it is an essential embryological process. Ducks, however, do
not exhibit cell death between the digits, and the result is webbing (syndactyly).

Slide 38: Review of proximodistal limb growth and anterior-posterior patterning

Main points:
1. FGFs initiate growth from the flank.
2. Expression of specific genes in the dorsal and ventral ectoderm establishes the AER.
3. SHH regulates anterior-posterior patterning.
4. FGFs secreted by the AER maintain the progress zone of proliferating cells.
5. As cells are left behind by limb outgrowth, they are distanced from the influence of
the AER, stop proliferating, and begin differentiating into cartilage.

Slide 39: Dorsoventral and bone patterning

Main points:
1. Dorsoventral (back of the hands versus the palms) is regulated by genes expressed in
the dorsal and ventral ectoderm.

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013
 2. Bone patterning is regulated by a complex pattern of overlapping HOX gene
expression.
Interesting point: Feedback loops exist between most of these molecular pathways so that the
overall limb pattern can be established.

Slide 40: Children with thalidomide-induced limb defects

Main points:
1. Thalidomide was used in the 1960s as a sedative and antinauseant mostly in Europe
and Australia. It did not get extensive use in the USA because the FDA was slow to
give its approval.
2. The drug interrupted function of the AER and proximodistal limb growth.
3. For some reason, digits often developed on short limbs (phocomelia), suggesting that
function of the AER was disrupted for a time, but then re-established.
4. Moms took the drug around the fourth to sixth weeks (at the time of limb
development).

Slide 41: Examples of polydactyly and brachydactyly

Main points:
1. In polydactyly, patterns of programmed cell death have been disrupted, leaving too
many parts of the AER.
2. In brachydactyly, something has caused the AER to stop working or the progress
zone to stop responding.

Slide 42: Examples of split foot and syndactyly (fused digits)

Main points:
1. Split foot is caused by excessive programmed cell death in either the AER or the
interdigital spaces (or both).
2. Syndactyly is an example of insufficient programmed cell death in the interdigital
spaces.

Slide 43: Examples of limb defects due to amniotic band syndrome

Main points:
1. Amniotic bands (strands of amniotic membrane) can wrap around limbs or digits and
cause amputations and rings.
2. The origin of bands is unknown. Perhaps fevers or viral infections alter the integrity
of the amniotic membrane, such that it can adhere to the conceptus and then tear.

Slide 44: More examples of amniotic band syndrome

Main points:
1. In addition to rings and amputations, bands can be swallowed causing severe facial
defects.

Sadler, T.W.: Langmans Medical Embryology, 12e Lippincott Williams & Wilkins, 2013