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CFB L5-10: Prenatal Craniofacial Development

Development of the pharyngeal apparatus I-II
Pharyngeal apparatus
Pharyngeal arches (aka branchial arches)
Pharyngeal pouches
Pharyngeal clefts
Pharyngeal arches
A series of tissue bulges that appear on the ventro-lateral side of the embryonic head
Transient structures, subsequently developing into the diverse components of the face and neck
 Transient structures that are only seen in early life and disappear with further development
A conserved feature in all vertebrate embryos
5 pairs of pharyngeal arches — 1st, 2nd, 3rd, 4th, 6th
5th arch degenerates immediately
 Some people don’t even think the 5th arch forms
Appear at the 4th and 5th weeks of gestation, in a rostral-caudal order
 First arch forms first and most rostrally; 6 th arch forms last and most caudally
 By the middle of the 4th week, we have the 1st and 2nd arches
 At then end of the 4th week, the 3rd arch has formed
 By the 5th week, the 4th arch has formed
st
1 pharyngeal arch = maxillary arch + mandibular arch
 Become the upper and lower jaw
Flatten and fuse later
 Arches flatten and fuse by the end of the 7th week, so the surface from head to neck is smooth
Pharyngeal clefts and pouches
Pharyngeal cleft = pharyngeal groove
 Indentation between pharyngeal arches on their external surface
 Lined by the ectoderm
 4 pairs of pharyngeal clefts
o 1st cleft is between the 1st and 2nd arches…4th cleft is between 4th and 6th arches
Pharyngeal pouches
 Indentation between pharyngeal arches on their internal surfaces
 Lined by endoderm
 4 pairs of pharyngeal pouches
o Pouches correspond to the same boundaries as the clefts, just on the inside when hemisected
Components of the pharyngeal arches
Epithelium
 Endoderm (inner surface, in pink)
 Ectoderm (outer surface, in blue)
Mesenchyme (under the epithelium)
 Neural crest-derived mesenchyme (just beneath the epithelium, in yellow)
 Mesoderm mesenchyme (at the core, in green)
Vascular endothelium (originates from mesoderm, all in purple)
 Pharyngeal (aortic) arch artery
o The only blood vessel you can identify at this stage
 Angioblasts
Nerves (in orange)
Components of the pharyngeal arch: Epithelium
Pharyngeal endoderm = wall of the pharyngeal gut
 At the rostral end of the foregut endoderm
 Covers the internal surface of the pharyngeal arches, and lines the pharyngeal pouches
 Becomes the lining of the pharynx and several glands in adults
Surface ectoderm
 Covers the external surface of the pharyngeal arches, and lines the pharyngeal clefts
 The ectoderm just outside of the pharyngeal arches form neurogenic placodes, which make sensory neurons innervating
the pharyngeal arches
 4 placodes that make neurons that contribute to the pharyngeal arches
o Trigeminal placode
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o Epibranchial placodes
 Geniculate placode
 Petrosal placode
 Nodose placode
Components of the pharyngeal arch: Mesenchyme
Neural crest derived mesenchyme
 Called ectomesenchyme or mesectoderm to distinguish it from the mesoderm mesenchyme
 Contains cranial neural cress cells (NCC) and cardiac NCC
 Cranial NCC:
o From forebrain (diencephalon), midbrain, and hindbrain
o Only those from the midbrain and hindbrain enter the
pharyngeal arches
 Cranial NCC from the forebrain go to more
frontoneural prominence
o Becomes skeleton and connective tissue (of the face and neck)
 Cardiac NCC:
o From mid-otic placode to the level of the 3 rd somite (2nd
pharyngeal cleft – 6th arch)
o Contribute to heart development
 Definitions based on where the NCC end up (retrospective) because you can’t really tell the difference between the
cranial/cardiac NCC after the otic placode
 NCC originating from the midbrain and hindbrain migrate in three streams
 The embryonic hindbrain is organized into 8 segments called rhombomeres (R)
 NCC from:
o Midbrain, R1, R2  1st pharyngeal arch
o R4  2nd pharyngeal arch
o R6, R7  3rd, 4th, and 6th pharyngeal arches
 R3 and R5 produce only very few CNCC, which join the streams from
neighboring rhombomeres
 First stream is cranial and cardiac NCC; 3 rd stream is cardiac NCC only!
Mesoderm mesenchyme
 Mostly from paraxial mesoderm, some from medial splanchnic mesoderm
 Paraxial mesoderm is divided into:
o Cranial/cephalic paraxial mesoderm — 1st, 2nd, and 3rd arches
 Upper area
o Somites (aka somites 1-5/occipital somites) — 4th and 6th arches
 Lower area
 Mesoderm core at the center of each pharyngeal arch
o Paraxial mesoderm, some splanchnic
o Mostly becomes skeletal muscles of the face and neck (branchiomeric muscles)
 Second heart field
o Splanchnic mesoderm
o At the junction of the pharyngeal arches and the heart
o Becomes a part of the heart (cardiac muscle)
 Cardiac and branchiomeric muscles are closely related due to shared origin (splanchnic mesoderm)
o Regulated by the same genes, thus a genetic defect in one will affect both areas
Components of the pharyngeal arch: Vascular endothelium
Pharyngeal (aortic) arch artery (PAA)
 PAA = aortic arch artery, aortic arch
 Embedded in the mesenchyme of the pharyngeal arches
 Branch off from the aortic sac to the dorsal aorta
 Derived from the splanchnic mesoderm
Angioblasts
 Precursors of blood vessels
 Dispersed throughout the pharyngeal arch mesenchyme
 From paraxial mesoderm and splanchnic mesoderm
Components of the pharyngeal arch: Nerves
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Cranial nerves
 Cranial nerves run through the pharyngeal arches
 Provide motor and sensory innervation to the pharyngeal arch derivatives
o 1st pharyngeal arch — trigeminal (CN V)
o 2nd pharyngeal arch — facial (CN VII)
o 3rd pharyngeal arch — glossopharyngeal (CN IX)
o 4th and 6th pharyngeal arches — vagus (CN X)
 Cell bodies of the neurons are outside of the pharyngeal arches
o Cranial nerves are in the pharyngeal arches, but their cell bodies are not
o Motor — coming from cranial motor nuclei in the hindbrain (rhombencephalon)
 Rostral-caudal order is maintained with the nerves going to motor nuclei
o Sensory — cranial ganglia
 Contain neurons from placodes and neural crest, glia from neural crest
 Position of ganglia are about the same as their placodes
 Placodes are thickening of endoderm — a surface structure
 Ganglia are not the same, they are internal structures
 Placodes are also transient structures that disappear, while ganglia are permanent
 Trigeminal ganglion is the only one that contains placode-derived and neural crest derived neurons
 You must remember the connection between each ganglion and nerve
Placode Ganglia Cranial Nerve
Ophthalmic lobe of trigeminal  trigeminal
Ophthalmic
ganglion
Trigeminal Trigeminal, CN V
Maxillomandibular lobe of trigeminal 
Maxillomandibular
trigeminal ganglion
Vestibulocochlear ganglion of CN VIII 
Geniculate Facial, CN VII
geniculate ganglion
Superior jugular ganglia of CN IX-X 
Epibranchial Petrosal Glossopharyngeal, CN IX
petrosal ganglion
Superior jugular ganglia of CN IX-X 
Nodose Vagus, CN X
nodose ganglion
Components of the pharyngeal arch
Various components of the pharyngeal arches from the same rostral-caudal position migrate coordinately
 E.g. Motor nerves coming from R4 contribute to the 2nd arch
 Neural crest cells derived from R4 go to 2nd arch
 Mesoderm cells lying next to R4 contribute to the 2nd arch
Pharyngeal arch development is regulated by interactions among the various components through secreted signaling molecules
Pharyngeal endoderm regulates development of the ectoderm and neural crest mesenchyme
 Pharyngeal endoderm is the master regulator
Sonic Hedgehog (SHH) is the major signaling molecule produced by the endoderm
Pharyngeal ectoderm regulates development of the neural crest mesenchyme
Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) are the major signaling molecules produced by the ectoderm
Derivatives of the pharyngeal apparatus
KNOW EVERYTHING ON THIS TABLE
Pharyngeal Arch Nerves Muscles Skeleton
1. Mandibular (maxillary V. Trigeminal: maxillary Mastication (temporal, Maxilla, zygomatic bone,
and mandibular and mandibular division masseter, medial and lacrimal bone, part of
processes) lateral pterygoids), temporal bone, palatine,
mylohyoid, anterior Meckel’s cartilage,
digastric, tensor veli mandible, malleus, incus,
palatini, tensor tympani anterior ligament of
malleus,
sphenomandibular
ligament
2. Hyoid Vii. Facial Facial expression Stapes, styloid process,
(buccinator, auricularis, stylohyoid ligament,
frontalis, platysma, lesser horn and upper
orbicularis oris, body of hyoid
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orbicularis oculi),
posterior digastric,
stylohyoid, stapedius
3. IX. Glossopharyngeal Stylopharyngeus Greater horn and lower
body of hyoid
4-6. X. Vagus Cricothyroid, levator veli Laryngeal cartilages
palatini, pharynx (thyroid, cricoid,
constrictors, intrinsic arytenoid, corniculate,
muscles of larynx cuneiform)
 *1st arch is sometimes called mandibular arch, but it contains maxillary and mandibular arches
Skeletal derivatives of the pharyngeal arches
Develop from the condensations of the mesenchyme
Bone that develops through intramembranous ossification
 Temporal, zygomatic, lacrimal, palatine, maxilla, mandible
 All come from the 1st pharyngeal arch
Cartilage
 Reichert’s cartilage — the cartilage of the 2nd pharyngeal arch
o Stapes, styloid process, stylohyoid ligament, lesser horn of hyoid, part of hyoid body
 Meckel’s cartilage degenerates — from the 1st pharyngeal arch
o Malleus, incus, and Meckel’s cartilage all from the 1 st arch
 Malleus, incus, and stapes (middle ear ossicles) undergo endochondral ossification, as do the styloid process and hyoid
o Malleus and incus are from the 1st arch; stapes is from the 2nd arch
 3 pharyngeal arch  greater horn of hyoid, part of hyoid body
rd

 4th pharyngeal arch  part of thyroid and arytenoid cartilages

 6th pharyngeal arch  part of thyroid and arytenoid cartilages, cricoid cartilage
All the skeleton from the pharyngeal arches is made of neural crest-derived cells (ectomesenchyme) EXCEPT arytenoid and cricoid
cartilage (lateral plate mesoderm)
Neuromuscular derivatives of the pharyngeal arches
Pharyngeal arch origin of each muscle in our face:
 Skeletal muscles develop from the mesoderm core of each pharyngeal arch
o 1st arch — mastication
o 2nd arch — facial expression
o 3rd arch — Stylopharyngeus
o 4th, 6th arches — soft palate, pharynx, larynx
As the muscle cells migrate, the nerves follow
 Note how widely distributed the muscles of facial expression are
 These muscle cells (especially from the 2nd arch) need to migrate very far to reach their destination
 The 2nd branchial arch is caudal to the 1st arch, but during maturation, the muscles from the 2 nd arch move more rostrally
than the muscles form the 1st arch
Vascular derivatives of the pharyngeal arches
Recall that the pharyngeal arch arteries (PAA) come from the aortic sac
PAA become parts of the great arteries in the adult
PAA undergo drastic remodeling to achieve the mature configuration of the great arteries
MUST KNOW THIS TABLE
PAA Right side Left side
1 Mostly disappear, maxillary artery Mostly disappear, maxillary artery
2 Mostly disappear, hyoid and stapedial arteries Mostly disappear, hyoid and stapedial arteries
3 Common carotid artery, internal carotid artery Common carotid artery, internal carotid artery
4 Right subclavian artery Arch of the aorta
6 Pulmonary artery Pulmonary artery, ligamentum arteriosum
 *Note how the left and right contributions are not always the same!
The endothelium of the pharyngeal arch arteries are surrounded by the tunic of vascular smooth muscle, which develops from
neural crest cells
Neural crest cells regulate remodeling of the pharyngeal arch arteries
 Perturbation of neural crest  malformation of the greater arteries
Contribution to the heart development from the pharyngeal arches
The heart is made of the cells from the 3 heart fields
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 First heart field (FHF = cardiac crescent), Second heart field (SHF), and Cardiac neural crest (CNC)
SHF and CNC cells localize to the pharyngeal region first, then move to the heart
Most of the heart comes from the 1st heart field, but the 2nd heart field has significant contribution as well
Cardiac neural crest cells contribute to the endocardial cushion and the septum of the cardiac outflow tract (OFT)
 Perturbation of neural crest  defective septation of OFT
 Truncus arteriosus and conus cordia are the only exit tube during development
 But eventually a wall/septum forms between the heart, so we get two tubes
 The outflow tract develops, and eventually consists of the aortic trunk and pulmonary trunk
Second heart field cells make the rest of the OFT, the right ventricle and parts of the atria
Derivatives of the pharyngeal clefts and pouches
The 2nd pharyngeal grows caudally over the 3rd and 4th arches, making a sealed cavity with the 3rd and 4th arches
 Thus, the 2nd, 3rd, and 4th arches close off and all get incorporated into the cervical sinus, which disappears later in
development
o 2nd arch will fuse with 6th week to close off access to the sinus, so it disappears
Pharyngeal cleft derivatives
 Cervical sinus (2nd, 3rd, 4th cleft)
 External auditory meatus (1st cleft)
Pharyngeal pouch derivatives
 Auditory tube, primitive tympanic cavity (1 st pouch)
 Palatine tonsil (2nd pouch)
 Inferior parathyroid gland, thymus (3rd pouch)
 Superior parathyroid gland, ultimobranchial body (4 th pouch)
Development of the glands from the pharyngeal apparatus
Parathyroid — from 3rd and 4th pouches
 Inferior parathyroid glands come from the 3 rd pouches;
Superior parathyroid glands come from the 4th pouches
 But the inferior parathyroid gland moves down past the superior parathyroid gland, so it’s position ends up being more
inferior
Thymus — from 3rd pouch
Ultimobranchial body — from 4th pouch
Thyroid – from the midline of the pharyngeal endoderm at the 2 nd arch level (foramen cecum)
 Thyroid is from the pharyngeal apparatus, but not the pharyngeal pouches (the pharyngeal endoderm instead); it migrates
caudally
 Parathyroid glands become attached to the thyroid once the thyroid has migrated down
Detach from the wall of the pharynx and migrate to the final positions
Thymus descends to the thorax, pulling inferior parathyroid parts of the way
Thyroid descends to the level of the first tracheal ring
Parathyroid glands become attached to the thyroid
Ultimobranchial body becomes a part of the thyroid
We get a connection between foramen cecum and the thyroglossal duct
Development of the tongue
Appear as 5 swellings in the beginning (4-5 weeks old)
 Two lateral lingual swellings and a tuberculum impar from the 1 st pharyngeal arch
 Copula (hypobranchial eminence) from the 2 nd, 3rd, and 4th pharyngeal arches
 Epiglottal swelling from the 4th pharyngeal arch
The body of the tongue forms from the two lateral lingual swellings (1 st arch)
The root of the tongue originates from the 2 nd (minor), 3rd, and 4th arches
 2nd arch almost disappears and is overpowered by the 3 rd and 4th arches
When refer to the body and root of the tongue, we are referring to the epithelial lining and connective tissue, but this does NOT
INCLUDE MUSCLES of the tongue
Mesoderm cells from the occipital somites invade to make tongue muscles
Branchial arches contribute to the epithelium and the connective tissue
Muscle cells migrate upwards to make the tongue muscles
Tongue muscles come from the somites, not the pharyngeal arches
Pharyngeal arches contribute to the epithelium and connective tissue

Birth defects involving the pharyngeal apparatus

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*Syndrome — a group of abnormalities occurring together

22q11 deletion syndrome

 Also called DiGeorge syndrome (broad definition)
 Caused by 1.5-3Mb of heterozygous deletion of chromosome 22q11.2 named based on the location In the chromosome
o Chromosome 22, location 11 on q arm
o Deletion of one copy of many genes in this area
 1 in ~3000 live birth
 Most common microdeletion syndrome
 Includes DiGeorge syndrome (DGS) and Velocardiofacial syndrome (VCFS)
 Characterized by defects in multiple derivatives of the pharyngeal apparatus
o Craniofacial malformation VCFS
o Cardiovascular abnormalities VSFC – DGS
o Deficiency in pharyngeal glands GDS
 Both diseases have some defects in all three areas (craniofacial, cardiovascular, and glandular)
 But if you see mild craniofacial phenotype and more severe cardiovascular and glandular phenotype, it is DGS; if you see
mild pharyngeal gland defect, and more severe cardiovascular and craniofacial abnormalities, it’s VCFS
TBX1 (T-box 1) is a key etiologic gene
 Encodes a transcription factor
 Expressed in the pharyngeal arches — ectoderm, endoderm, mesoderm (regulates neural crest derived cells indirectly via
intercellular signaling)
 Not expressed in neural crest derived cells
 If you knock out TBX1, you’ll see it expressed in neural crest derived cells
1. Craniofacial phenotypes not always all the symptoms are in one patient – these are the common
- Small lower jaw (micrognathia), small mouth, square nasal tip
- Low-set ear, abnormally folded pinna, middle ear abnormality
- Spectrum of palate defects (submucous over cleft, high arched palate, bifid uvula (mild manifestation of cleft palate)).
- Mostly explained by impaired development of the structures derives from the 1st and 2nd pharyngeal arches
2. Cardiovascular phenotypes
- Abnormal great arteries — interrupted aortic arch (IAA), aberrant right subclavian artery (A-RSA)
o IAA — break in aortic arch, which should be continuous
o A-RSA — right subclavian artery becomes directly attached to the aortic arch (instead on attaching to common
carotid artery)
o Caused by developmental defects in the 4th pharyngeal arch artery (arch of aorta and RSA are derived from the 4 th
pharyngeal artery)
- Heart defects — persistent truncus arteriosus (PTA), transposition of the great arteries (TGA)
o PTA - truncus arteriosis fails to properly divide into pulmonary trunk and aorta
o TGA — aortic and pulmonary separation doesn’t happen in a spiraling way, so they just go straight up/down
(instead aorta from the LV it comes out from the RV and same with pulmonary artery from LV instead of RV)
o Disruption in development (failure, aberrant formation) of the cardiac outflow tract septum (from cardiac neural
crest cells)
o These patients will have 1 OFT instead of 2 — they maintain the embryonic structure of the heart
o Defect explained by cardiac neural crest cells because they contribute the most to the cardiac OFT
3. Deficiency in the pharyngeal glands
1- Hypocalcemia (low serum calcium levels; might cause seizures)
o Lack of parathyroid hormone due to impaired/failed development of the parathyroid glands
2- Immunodeficiency (recurrent ear and respiratory infections)
o Lack of, or impaired function of T cells due to impaired development of the thymus glands
 Caused by developmental defects in the 3rd pharyngeal pouch (gives rise to inferior parathyroid and thymus)

Treacher Collins syndrome

AKA Mandibulofacial Dysostosis
1 in ~10,000 births
1) Disorder of craniofacial development (mostly 1st pharyngeal arch)
2) Caused by craniofacial, cardiovascular, and glandular defects, but mostly due to craniofacial
3) Micrognathia, hypoplastic zygomatic arches, macrostomia (wide mouth), and cleft palate
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4) Mostly affects the jaw region

5) Slanted eyes, lower eyelid coloboma
6) Microtia (small looking ears) and other deformity of the ears, Deafness
 Both 22q11 and treacher collins syndrom have micrognathia, but 22q11 causes narrow mouth and treacher collins
syndrome causes wide mouth  BIG DIFFERENCE BETWEEN THE TWO
 Defining feature is the craniofacial defects
o Caused by heterozygous mutation in TCOF1, encoding the Treacle protein
o Treacle is involved in making ribosomes (rRNA production)
o TCOF1 is expressed in the neuroepithelium and neural crest cells
o TCOF1 haplo-insufficiency leads to deficiency in number of neural crest cells due to increased cell death and decreased cell
proliferation decrease in number of neural crest cells
 Haplo-insufficiency = deletion of one gene, which In some genes  insufficient for development

(Pierre) Robin sequence (syndrome)

 Sequence — one cause leading to a chain of developmental malformations
o One type of syndrome
o Must have a causal relationship in order to be a sequence
 1 in ~8500 births
 Disorder of craniofacial development (mostly 1 st pharyngeal arch)
 Micrognathia (small lower jaw), glossoptosis (downward displacement or retraction of the tongue), cleft secondary palate
(each lead to the next  sequence) you have the three characteristics
o Oral cavity is so small that the tongue has nowhere else to go so it goes down/retracts
o There’s a likely causal relationship between microagnathia  glossoptosis  cleft secondary palate
 This IS the sequence
 Leading to life-threatening upper airway obstruction  respiratory distress and feeding difficulties (can’t suckle properly)
 Genomic locations in Chr17 and Chr2 have been associated with PRS
 Candidate genes include SOX9 (in Chr17), a transcription factor that is essential for skeletal development

Goldenhar syndrome
= Oculoauriculovertebral spectrum, Hemifacial macrosomia
 Hemifacial macrosomia = asymmetric phenotype; only affects one side
o Half of the face is small
 Under-development of the oral-facial area (zygomatic arch, palate, mandible, lips), nose, eye, ear, and vertebrae usually on
one side only
 Abnormal development of the 1st and 2nd pharyngeal arches
 May be accompanied by defects in other organs (heart, kidney, lung, brain…)
 Cause unknown

Branchial fistulas and lateral cervical cyst not a syndrome

 Aberrant development of the pharyngeal clefts
 Normally the 2nd, 3rd, and 4th pharyngeal clefts are covered up by the 2nd pharyngeal arch, become the cervical sinus, and
then disappear
 Branchial fistulas (external) occur when the 2nd pharyngeal arch fails to grow caudally over the 3 rd and 4th arches
 Fistula (pipe) keeps the 2nd, 3rd, and 4th clefts in contact with the surface
 Internal branchial fistulas (rare) result from the rupture of the membrane between the 2 nd cleft and 2nd pouch
o Thus they form a connection
 Branchial fistulas provide drainage for a lateral cervical cyst that failed to disappear
o Without drainage, the cyst continues to persist and grow
 Branchial fistulas and lateral cervical cysts are found directly anterior to the sternocleidomastoid muscle

Thyroglossal cyst and fistula

 During migration of the thyroid gland from the foramen cecum, a canal forms connecting the two (thyroglossal duct)
 Thyroglossal duct normally disappears (in normal cases there is no connection between foramen cecum and thyroid gland)
o If not  thyroglossal cysts (due to accumulation of fluid)
 Thyroglossal cysts can rupture if there is too much fluid, creating a fistula connecting to the outside
 Thyroglossal cysts may lie at any point along the migratory path of the thyroid gland
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 Always close to the midline

o Cysts are formed at the midline and go down the neck

Tongue tie (ankyloglossia)

 Tongue is not freed from the floor of the mouth due to the frenulum at the tip of the tongue
o Short attachment of the lingual frenum

Development of the face, lip, palate, and related birth defects

Facial prominences
 Bulges of epithelium-covered mesenchyme that form in the presumptive facial region of an embryo
o Facial prominences = frontonasal mass (FNM) + 1 st pharyngeal arch (PA1 or BA1, 1st branchial arch)
o PA1 = maxillary arch (mxPA1) + mandibular arch (mdPA1)
o FNM = lateral nasal prominence (LNP) + medial nasal prominence (MNP)

Development of the face

 At the end of the 4th week, 5 facial prominences surround the stomodeum
 Frontonasal prominence (FNP), a pair of maxillary prominences (MXP), a pair of mandibular prominences (MDP)
 A pair of nasal placodes form on FNP
o Nasal placodes form your nose; eventually invaginate to form the nostrils

Prominence contribution to the face

KNOW THIS TABLE
Prominence Structures formed
Medial nasal Philtrum, bridge, crest, and tip of the nose; mesial portion of the upper lip; incisors
Lateral nasal Alae of the nose
Maxillary Cheeks; lateral portion of the upper lip; canines, premolars, and molars
Mandibular Lower lip; chin; lower jaw

Development of the face

5 weeks
 Nasal placode  nasal pit
 Nasal prominences (lateral, medial)
 Maxillary prominence grows medially, compressing the medial nasal prominence
7 weeks
 Medial nasal prominence and maxillary prominence fuse  upper lip
 Lateral nasal prominence does NOT participate in lip development
o No contribution of lateral nasal prominence in formation of the lip
 Lower lip forms from the fusion of the two mandibular prominences
 Nasolacrimal groove — between lateral nasal and maxillary prominences
o Gives rise to the lacrimal sac and nasolacrimal duct (eye-nose)
Development of the nose
The middle part of the nose develop from the Medial nasal prominance
Medial nasal prominence gives rise to the philtrum of the upper lip, middle part of the nose
 Epithelial seam between the medial nasal and maxillary prominences must disappear (5-7 weeks) to give rise to the upper
lip
 Epithelial seam is removed by
o Apoptosis
o Epithelium-to-mesenchyme transition
Lateral nasal prominence gives rise to the alae of the nose

Development of the primary palate

Intermaxillary segment
 Forms from the fusion of the two medial nasal
prominences
 Gives rise to the philtrum of the upper lip, median part of
the maxillary bone with 4 incisors, and the primary palate
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 Primary palate (yellow, intermaxillary segment) and secondary palate (red) form from different embryonic structures and at
different times
 Primary palate is more anterior than the secondary palate


Development of the secondary palate

 From the fusion of the palatal shelves (outgrowth on the medial side of the maxillary arches)
 More complex than the primary palate, and formed later
 Uvula is at the end of the secondary palate
 Stages of secondary palate development
1. Formation of the palatal shelves — 6 weeks
2. Vertical elongation — 6.5 weeks
3. Elevation — 7.5 weeks
4. Horizontal growth
5. Fusion (disappearance of the epithelial seam) — 10 weeks
 Get cleft palate if any of these steps go wrong
o Thus many gene defects can cause left palate!
 Palatal shelves initially grow downward on either side of the tongue
 Epithelial-mesenchymal signaling interactions involving secreted molecules are essential
o FGF — fibroblast growth factor
o BMP — bone morphogenic protein
o SHH — sonic hedgehog
 Palatal shelves elevate above the tongue to a horizontal position
 Two mechanisms of palatal elevation — quick flip, gradual remodeling
o Quick flip — anterior part of the palate
o Gradual remodeling — posterior part of the palate
 Cells move and make a little bulge, then flip
o Regulation of these two events is unknown
 Palatal shelves meet at the midline to form the medial epithelial seam (MES)
o MES disappears to complete the fusion between the palatal shelves
o Fuse with primary palate anteriorly, and with nasal septum dorsally
 What happens to the cells of the MES?
o MES cells get pushed outward
o Displaced and extruded from MES to oral and nasal surface of the palatal shelf
o Undergo apoptosis

Ossification of the palate

 Primary palate — hard palate
 Secondary palate — hard palate + soft palate
o The boundaries are not what we are inclined to think they are
o Secondary palate doesn’t start at the boundary between hard and soft palate, it includes a portion of hard palate

(Oro)facial clefts — cleft lip and cleft palate

 Caused by the partial or complete lack of the fusion between the facial prominences
 Among the most common congenital malformations
 1 in ~700 births — very high frequency for birth defects!
 Frequency varies by race, ethnicity, gender
o Native Americans, Asians > Caucasians > Africans
 Can be categorized multiple ways
o CL/P (cleft lip with or without cleft palate) vs. CPO (cleft palate only)
 Cleft lip, cleft palate (CL/P) — lack of fusion between the medial nasal and maxillary prominences
(epithelial seam)
 Cleft secondary palate (CPO) — lack of fusion between the palatal shelves (medial epithelial seam)
 Can only get CPO (without cleft lip) for secondary palate
o Isolated (non-syndromic) > Syndromic (multiple defects occurring together, e.g. cleft lip/palate with heart and/or
kidney defects)
o Unilateral vs. Bilateral — cleft on one vs. both sides
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o Complete vs. Incomplete — facial prominences/tissue not touching at all vs. touching somewhat
o Mucous vs overt
 Mucous cleft —  when you can’t see the cleft from the outside because the epithelium on the outside is
fused, but the internal structures are not
 Overt cleft — a cleft that is visible from the outside
 3 major birth defects overall:
1) Oro-facial clefts
2) Cardiac outflow tract defects
3) Neural tube defects
Some common examples of cleft lip/palate
 Unilateral cleft lip (partial, incomplete)
 Complete bilateral cleft lip
 Complete cleft secondary palate (CPO)
 Complete bilateral cleft lip and the primary palate, and complete cleft secondary palate
Orofacial clefts — rare types
 Midline cleft of the mandible — lack of fusion between the two mandibular prominences
 Median cleft of the upper lip — lack of fusion between the medial nasal prominences
o A form of frontonasal dysplasia
 Oblique facial clefts — lack of fusion between the maxillary and lateral nasal prominences

Why orofacial clefting happens during development

 Prominences (palatal shelves) are deficient in shape/size and don’t make contact
 Prominences (palatal shelves) contact but too late in development
 Prominences (palatal shelves) make contact but epithelium is not removed
 Tongue interferes with the elevation of the palatal shelves
And many other potential reasons…

Etiology for CL/P is multifactorial

Genetic:
 Genes associated with CL/P in humans:
o TBX22, IRF6, MSX1, PVRL1, P63, CDH1, EFNB1, TGFB3
 Interaction of many genes of small effect can produce significant phenotypic deficiencies
Environmental factors:
 Nutrition, teratogens (alcohol, smoking, etc)

Development of the CNS and related birth defects

CNS development in an early embryo
Neural plate
Neural groove and folds
Neural tube
Recall, neural plate  neural folds
 Neural grooves are found in between the neural folds
Neural folds will fuse to form the neural tube
 Fuses first in the center, then zips up rostrally/caudally
 By the 4th week, the neural tube is almost finished closing, except cranial and caudal neuropores
 Body axis is still straight
After the neuropores close, we get the 3 primary vesicles of the brain
Three vesicle stage — 4th week of gestation
 CNS starts to bend here
 Prosencephalon (forebrain), mesencephalon (midbrain), rhombencephalon (hindbrain)
Five vesicle stage — 5th week of gestation
 Prosencephalon  telencephalon, diencephalon
 Mesencephalon (does not divide)
 Rhombencephalon  metencephalon, myelencephalon
Signaling centers regulating brain development
Signaling centers are specific parts of the tissue that produce specific signaling proteins; organizing centers
Prechordal plate — mesoderm anterior to the notochord
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 Produces SHH
 Regulates development of the midline structures of the brain and face
Anterior neural ridge — the most anterior region of the neural plate, bordering the non-neural ectoderm
 Produces FGF
 Regulates development of the telencephalon
Isthmus — border between the midbrain and hindbrain
 Produces FGF and WNT
 Regulates development of the midbrain and hindbrain
Development of the cerebral hemispheres
Pallium develops into the cerebral cortex
Subpallium gives rise to basal ganglia
Start to get massive outgrowth of the telencephalon after 5 weeks, that covers
up the rest of the brain
Cross section of the telencephalon
 Pallium is a thin layer of tissue on the top/dorsal part
 Subpallium is beneath the pallium
 These layers start thin, and get thicker as the brain grows
 Corpus striatum is a bulge of tissue growing within the subpallium
 Subpallium gives rise to the corpus striatum, which gives rise to the basal ganglia
In order for the pallium and subpallium (or brain) to grow, we need more cells
Ventricular zone — contains the proliferating neuronal progenitors
Differentiating neurons migrate away from the ventricular zone via radial migration
 Radial migration — cells from the pallium migrating into the cerebral cortex
Some neurons from the basal ganglia migrate to the pallium via tangential migration and become a part of the cortex
 Tangential migration — cells from the striatum migrating into the cerebral cortex
Thus, two types of migrations contribute to the massive growth of the cortex
Neural tube defects (NTD)
Neural tube fails to close properly in the 4th week of development
1 in ~1500 births in the US
Children with severe neural tube defects prone to hydrocephaly (related to Arnold-Chiari malformation)
50%-70% of all NTDs can be prevented by maternal use of folic acid (folate, vitamin B9, 400µg/day) prior to and during pregnancy
Spina bifida
Failure in fusion of the bony arch of one or more vertebrae
Spina bifida without neural tube defects:
 Spina bifida occulta — the bony defect is covered by skin;
the spinal cord is intact
o Does not result in disability
 Spina bifida cystica — protrusion from outside the body
o Meningocele — fluid filled sac of meninges
protrudes from the defect
o Meningomyelocele — neural tissue and meninges protrude
Spina bifida with neural tube defects:
 D and E Rachischisis (spina bifida with myeloschisis) — neural tube fails to close, resulting in spina bifida
o Neural tissue becomes necrotic
o Most severe type!
Arnold-Chiari malformation
 Found in almost all cases of spina bifida cystic
 Caudal displacement and herniation of part of the cerebellum through the foramen magnum
o Cerebellum is pushed into foramen magnum
o Central canal is blocked so CSF is stuck in the brain area and cannot go into the spinal cord
 Obstructs the flow of CSF and thus causes hydrocephalus
o Hydrocephalus — excessive buildup of CSF in the ventricles
How spinal bifida cystica leads to Arnold-Chiari malformation
 During normal development, the vertebral column elongates faster than the spinal cord
 Because the spinal cord is not attached to the vertebral column, each segment of spinal cord slides up
with respect to the corresponding segment of vertebral column
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 In the case of spina bifida cystica, the cyst acts as a knot, anchoring the spinal cord to the vertebral canal at that position
 As the vertebral column elongates, the portion of spinal cord between foramen magnum and the cyst gets stretched
 This stretch creates the force for the spinal cord to pull the cerebellum down into foramen magnum
Neural tube defects of the brain
Exencephaly — “open brain”
 As a result, the vault of the skull does not form
 Leads to anencephaly
Anencephaly — “no brain”
 Brain neural tissue is dead and degraded (brain stem spared)
 Brain tissue is exposed to fluid around the embryo, which has enzymes that break down the brain tissue
Craniorachischisis — NTD in brain and spinal cord
 Leads to anencephaly as well as defect involving the spine
Meningoceles, meningoencephaloceles, and meningohydroencephaloceles
 Neural tube closure defect  ossification defect in bones of the skull
o Brain develops first, so if there’s something wrong with the brain, bone just won’t grow over it
 The most frequently affected bone is the occipital bone
 Meningocele — if the opening of the occipital bone is small, only meninges bulges through it
 If the defect is large, part of the brain (menigoencephalocele) and even part of the ventricle (meningohydrocephalocele)
penetrate through the opening into the meningeal sac
 Most can be prevented by maternal use of folic acid (400µg/day)
o Embryonic origin of these defects are from neural tube closure defects


Cranial defects
Holoprosencephaly (DPE) — a spectrum of abnormalities in which a loss of midline structures results in malformation of the brain
and face
 1 in 15000 live births, but 1 in 250 pregnancies that end in early miscarriage
 Alobar HPE — severe form; one telencephalic vesicle, one eye, one nasal chamber, along with other midline facial defects
o Alobar = no lobe  I.e. no distinction between the two lobes, only one vesicle
 Lobar HPE — less severe form; some division of the prosencephalon in two cerebral hemispheres
o Incomplete development of midline structures (a midline cleft lip, lack of nasal tissue, hypotelorism)
 Very mild cases possible — e.g. presence of a single central incisor
 Mutation sin the SHH gene is one cause of HPE
 Recall that SHH dictates development of midline structures of the brain and face (CNS)
o Prechordal plate produces SHH, which regulates development of midline structures
Smith-Lemli-Opitz syndrome
 Autosomal recessive condition
 Due to mutations in a gene for 7-dehydrocholesterol reductase (DHCR7), which metabolized 7-dehydrocholesterol to
cholesterol
 Defects in a variety of organs including the face (micrognathia, cleft palate, broad flat nose), eye and ear (ptosis, low set
ears), heart, brain, limbs, etc
o 5% have HPE
 May be due to abnormal SHH signaling
 Cholesterol is essential for SHH activity
o Cholesterol is attached to the part of the SHH protein that is cleaved, and this attachment is essential
Schizencephaly
 A rare disorder in which large clefts occur in the cerebral hemispheres, sometimes causing a loss of brain tissue
o Tissue is unable to develop
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 Developmental delay, cerebral palsy, or seizure

 Mutations in the homeobox gene, EMX2 (a transcription factor)
 May be caused by vascular disruptions during fetal brain development
Hydrocephaly
 Abnormal accumulation of CSF within the ventricular system
 In most cases, due to an obstruction of the cerebral aqueduct (aka aqueduct of Sylvius)
o Aqueductal stenosis — obstruction of the aqueduct
 Can also be caused by Arnold-Chiari malformation
 As a result, fluid accumulates in the lateral and third ventricles and presses on the brain and bones of the skull
 Because the cranial sutures had not closed, pressure from the accumulated fluid enlarges the head, thinning the bones of
the skull and cerebral cortex
Microcephaly
 The cranial vault that is smaller than normal
o Top part of the head containing the brain is very small compared to the rest of the face
o Brain is smaller, so the face looks bigger in proportion
 Because the size of the cranium depends on growth of the brain, the underlying defect is poor growth of the brain
 Causes are variable; genetic, infection or exposure to teratogens
 Frequently associated with intellectual disability
 How does brain size determine cranium size?
o Sutures contain progenitors of osteoblasts
 As the brain grows, the suture area has undifferentiated mesenchyme that produces osteoblasts to
produce more bone
 Thus the suture area stretches out, causing mechanical pressure
o Mechanical pressure stimulates these progenitors to proliferate and differentiate to make more bone

Development of the eye and ear, and related birth defects

Development of the ear
The ear has both auditory and vestibular functions
External ear — collects sound
Middle ear — conducts sound from the external to the internal ear
Internal ear — converts sound waves into nerve impulses and registers changes in equilibrium
Development of the internal (inner) ear)
Otic placodes – appear at ~22 days
 Invaginate to form the otic vesicles (aka auditory vesicles, otocysts)
 Otic placode invagination  otic pit  otocysts
Otocyst divides into:
 1. Ventral component — saccule and cochlear duct
 2. Dorsal component — utricle, semicircular canals, and endolymphatic duct  membrane labyrinth
1. Development of the scala vestibule and scala tympani
 Mesenchyme surrounding the cochlear duct differentiates into cartilage  Vacuolization (10th week)  perilymphatic
spaces, the scala vestibuli and scala tympani
o Vacuolization — cartilage develops holes inside
 Vestibular membrane — between the cochlear duct and scala vestibuli
 Basilar membrane — between the cochlear duct and scala tympani
2. Development of the organ of corti
- Cochlear duct  inner ridge and outer ridge
o Outer ridge — forms hair cells, covered by the tectorial membrane (= organ of corti)
3. Utricle  semicircular canals (5-8th week)
- Flat part undergoes morphogenesis to become the shape of the semicircular canals
Summary of steps
- Otocyst from the otic placode elongates, undergoes morphogenesis, and gives you the cochlear duct, utricle, and
semicircular canals
- Take home: the whole inner ear comes from the otocyst!
Development of the middle ear
1. Tympanic cavity and auditory tube (eustachian tube)
- Both from the 1st pharyngeal pouch
2. Ossicles
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- From neural crest derived mesenchyme of the 1st pharyngeal arch (incus, malleus) and the 2nd pharyngeal arch (stapes)
o Develop as cartilage and then undergo endochondral ossification afterwards
o Form from mesenchyme condensation
Development of the external ear
1. External auditory meatus
- From the 1st pharyngeal cleft
2. Eardrum (tympanic membrane)
- Made from all 3 germ layers
- The eardrum is made up of (1) an ectoderm lining at the bottom of the auditory meatus, (2) an endoderm epithelial lining of
the tympanic cavity, and (3) a layer of connective tissue from mesoderm
3. Auricle (pinna)
- Develops from auricular hillocks — 6 mesenchymal proliferations at the dorsal ends of the 1 st and 2nd pharyngeal arches,
surrounding the 1st pharyngeal cleft
- As fusion of the auricular hillocks is complicated, developmental abnormalities of the auricle are common
- Fusion and elongation of clefts/arches  formation of the structure
Congenital deafness
1 in 1000 – 2000 births
Mostly autosomal recessive
Non-syndromic more common than syndromic
Caused by genetic and environmental factors
- Connexin genes (gap junction)
- Rubella and cytomegalovirus infections during pregnancy
- Maternal diabetes
Mondini malformation (dysplasia) — cochlea fails to make spiral turns
External ear defects
All of the frequently occurring chromosomal syndromes have ear anomalies as one of their characteristics
- Microtia — ear is smaller than normal
- Preauricular appendages: skin tags – accessory auricular hillocks
- Preauricular pits: shallow depressions – abnormal development of the auricular hillocks
Eye development
1. Optic cup and lens vesicle
- Optic groove  optic vesicle
o Optic vesicle develops from the diencephalon neuroepithelium
- Optic vesicles induce the surface ectoderm to make the lens placode
o Lens placode  lens vesicle
- Optic vesicle  optic cup
o Optic vesicle invaginates further to form the optic cup
- Invagination of the inferior surface of the Optic Cup forms the Choroid Fissure
- Choroid fissure allows the hyaloid artery to reach the inner chamber of the eye
- During the 7th week, the lips of the choroid fissure fuse
- Lens placode invaginates  lens vesicle
- Lens vesicle loses contact with the surface ectoderm
2. Retina
- From the optic cup
o Outer layer  pigmented layer of the retina
o Inner layer  neural layer of the retina
3. Iris
- Iris develops from the tip of the optic cup
- The sphincter and dilator pupillae muscles develop from the ectoderm of the optic cup
o Note, most muscles develop from the mesoderm!
4. Lens
- From the lens vesicle
o Cells of the posterior wall elongate and form long fibers that fill the lumen of the vesicle
- By the end of the 7th week, these primary lens fibers reach the anterior wall of the lens vesicle
5. Choroid, sclera, and cornea
- At the end of the 5th week, the eye primordium is completely surrounded by loose mesenchyme
o Inner layer  choroid
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o Outer layer  sclera

6. Optic nerve
- Inside optic stock, along with a portion of the hyaloid artery (later called the central artery of the retina)
- On the outside, a continuation of the choroid and sclera, the pia arachnoid and dura layer of the nerve respectively,
surround the optic nerve
- Start forming the optic nerve by week 6, and finish by week 9
SHH from the prechordal plate splits the eye field into two
Perturbation of SHH signaling can lead to cyclopia (one eye)
PAX6 — a key regulator of eye development
SHH from prechordal plate positively regulates PAX2, represses PAX6 from the midline
PAX2 regulates optic stock differentiation
Coloboma
A hole in one of the structures of the eye, such as the iris, retina, choroid, or optic disc
If choroid fissure fails to close  coloboma in the iris
Can also have coloboma in the eyelid