NEURULATION AND CRANIO-FACIAL DEVELOPMENT LEARNING OUTCOMES 1. Describe the induction of the neural plate by the notochord and the progressive formation of the neural tube 2. Explain the origin of the neural crest cells, their migration and eventual destinations 3. Show the segmental pattern of nerve development in the spinal cord and the relationship between nerve, and muscle derived from the myotome 4. Outline the segmentation of the brain 5. Describe the congenital malformation of the nervous system, e.g. spinal bifida, cerebellar hypoplasia and hydrocephalus. 6. Outline the development of the nasal passage and the mouth 7. Understand the development of the ear and the eye

The notochord induces the overlying neuroectoderm cell layer to invaginate to form the neural tube Represents paracrine signals Neural tube closure begins near the rostral end of the embryo and progresses caudally 1. Cells near the midline of the ectodermal layer elongate to form the neural plate, induced by the proximity of the notochord, a rod of mesodermal cells. 2. The neural plate folds inwards and seals to form the neural tube. 3. Closure begins at the upper spinal cord and then progresses forwards and backwards until only the anterior and posterior neuropores remain (these close later). 4. Neural crest cells originate at the junction with the ectoderm where the neural tube closes and they migrate to diverse destinations and take on diverse identities (see later).

2. The neural plate folds inwards and seals to form the neural tube. Neuro-epithelial layer already showing signs of elongation of cells (notochord not present) Neural groove forming 1. Cells near the midline of the ectodermal layer elongate to form the neural plate, induced by the proximity of the notochord, a rod of mesodermal cells. 2. The neural plate folds inwards and seals to form the neural tube. 3. Closure begins at the upper spinal cord and then progresses forwards and backwards until only the anterior and posterior neuropores remain (these close later). 4. Neural crest cells originate at the junction with the ectoderm where the neural tube closes and they migrate to diverse destinations and take on diverse identities (see later). Neural tube formed with overlying ectoderm and underlying notochord

The neural fold closes from a starting cervical location in both a rostral and caudal direction http://www.med.unc.edu/embryo_images/ VENTRAL VIEW Heart Foregut Hindgut Non-fused neural folds Mouse 8 days DORSAL VIEW Mouse 9 days Anterior neuropore Mouse 9 days caudal neuropore

Neural crest cells escape the neuroectoderm epithelium and migrate to diverse destinations Neural crest cells leaving dorsal ectoderm (Gilbert) (Epithelial to mesenchyme transition) 1. Neural crest cells originate at the junction with the ectoderm where the neural tube closes and they migrate to diverse destinations and take on diverse identities. 2. Neural crest cells give rise to the neurones of the dorsal root ganglia [2] 3. The trunk neural crest cells also give rise melanocytes [1], neurones of sympathetic ganglia [3], the neurone like cells of the adrenal medulla[4] and the neurones of the submucosal nerve plexus of the gut.[5] Neural crest cells escape the neuroectoderm epithelium and migrate to diverse destinations4. In the process, neural crest cells must undergo an epithelial to mesenchymal transition and penetrate a basal lamina.

Neural Crest Cell Induction FoxD3, Slug Wnt6, ectoderm BMPs Ectoderm Neural crest precursors Neural tube

Neural crest migration Slug activates factors inducing the dissociation of tight junctions Migrating cells follow cues from the extracellular matrix One set of proteins (fibronectin, laminin) promote migration while ephrin impedes migration (remember lectures on cell adhesion and control of cell division)

The spinal cord develops a segmentation pattern which reflects the pattern of somites. SEGMENTATION IN THE SPINAL CORD AND PERIPHERAL NERVES PIG - 38 DAYS PIG - TERM Dermis Muscle A segmental reflex arc Sensory neurone Inter- neurone Dorsal root ganglion Dorsal horn Ventral horn Motor efferent 1. The spinal cord develops a segmentation pattern which reflects the pattern of somites. 2. The ventral roots of the spinal cord are formed by the production of axons by neuroblasts of the neural tube and these connect to muscles to form motor nerves 3. The dorsal root is formed by neuroblasts in adjacent sensory ganglia (these arising from neural crest cells) which produce axons which link the grey matter of the spinal cord via the dorsal root with sensory locations in the body

Dorsal-ventral axis in the Neural Tube TGFb family in ectoderm TGFb family in roof plate Gradient of TGFb family Interneurones Gradient of shh Motor neurones shh in notochord shh in floor plate shh 1. The notochord produces Sonic hedgehog (Shh) and induces the ventral neural tube to become floor plate and produce Shh 2. The ectodermal cells produce members of the Transforming growth factor (TGF-b) family and induce the dorsal neural tube to become roof plate and to start to produce the same proteins 3. Two gradients are created of TGF-b and Shh 4. Different concentrations of these proteins activate the expression of different sets of genes so that cells differentiate to become inter-neurones and motor neurones

The head also shows a rostral/caudal segmentation pattern but this is less regular and more complex than that of the trunk somites 1. The head and branchial regions also show evidence of segmentation but in a much less regular way and in a way that is distorted by subsequent embryonic movements and remodelling. 2. The brain itself passes through a 3 vesicle stage with fore, mid and hind brain to a 5 vesicle stage in which the fore and hindbrain are further subdivided. Based on Noden and LaHunta p171, p100

SEGMENTATION OF THE HEAD REGIONS OF THE BRAIN Di- Tel- The forebrain gives rise to the Tel- and Di-encephalon vesicles Mes- The midbrain gives rise to the Mes-encephalon vesicle Met- Myel- The hindbrain gives rise to the Met- and Myel-encephalon vesicles Faint evidence of rhombomere segmentation of met- and myel-encephalon Mouse 10 days. http://www.med.unc.edu/embryo_images/

The branchial arches are bilateral pouches of tissue separated by branchial clefts in the region of the pharynx Arch 1: maxillary Arch 1: mandibular Arch 1: maxillary Arch 2 Arch 3 Arch 1: mandibular Arch 2 Mouse, 10 days, lateral view Human, 30 days, ventro-lateral view http://www.med.unc.edu/embryo_images/

The branchial arches are separated internally by pharyngeal pouches and externally by branchial clefts Mid-brain and vesicle MIDLINE Oral cavity Torn edge of oral plate Floor of pharynx 1 2 3 Branchial arch Pharyngeal pouch Branchial cleft Laryngo- Tracheal groove AORTIC ARCH Mouse, 9 days, section, from dorsal view http://www.med.unc.edu/embryo_images/

The branchial arches and clefts and the juxtaposed pharyngeal pouches are a recapitulation of the respiratory anatomy of fish 1. Certain cranial nerves innervate the branchial arches and the adult derivatives of these. 2. Between the branchial arches are the branchial clefts which align with diverticula of the pharynx called the pharyngeal pouches. 3. In fish, there are 6 pharyngeal pouches and associated branchial cleft s and these become continuous to form the external gill arches. 4. In mammals and birds this fusion does not take place and only the first 3 or 4 branchial arches, clefts and pharnygeal pouches are prominent.

There are 12 cranial nerves corresponding to the 7 somitomeres and 5 rostral somites of the head region 1. The 7 cranial somitomeres and the 5 most anterior somites are matched by 12 pairs of cranial nerves which originate in different regions of the brain. 2. The olfactory (I) and optic (II) nerves are brain tracts rather than true nerves 3. The cranial nerves usually do not unite the dorsal and ventral roots and may be either only motor, only sensory or mixed.

Motor cranial nerves follow their corresponding myotome to find their adult path SEGMENTATION OF THE HEAD - CRANIAL NERVES, MOTOR EFFERENTS AND TARGET MUSCLES 5 SOMITES XII XI X IX VII VI V IV III 1 2 3 4 5 6 7 7 SOMITOMERES TONGUE CRANIAL NECK MUSCLES LARYNGEAL MUSCLES PHARYNGEAL MUSCLES FACIAL MUSCLES EYE MUSCLES MUSCLES OF 1. The cranial efferent nerves emanating form the regions of the mid and hind brain initially contact unsegmented somitomeres or segmented somites closest to them. 2. What complicates the later picture is that muscle primordia originating in the somitomeres or the somite myotome migrate to new destinations (see arrows). 3. Thus cranial nerves III, IV and VI grow along with the somitomere-derived cells that form the eye muscles. 4. Cranial nerves V,VII,IX and X project to muscle primordia that enter the branchial arches. V innervates the muscles of mastication, VII the muscles of facial expression, IX the muscles of the pharynx and X the muscles of the larynx. 5. XI innervates the superficial muscles of the shoulder neck and head which are believed to be homologues of the gill levator muscles in fish. 6. XII arises from several roots and innervates the muscles of the tongue. 7. Sensory afferents carry information on touch (V), vision (II), olfaction (I), taste (VII,IX and X) and hearing and vestibular senses (VIII). MASTICATION ROMAN NUMERALS ARE THE CRANIAL MOTOR NERVES ARROWS FROM SOMITOMERES AND SOMITES INDICATE THE MIGRATION OF THE MYOBLASTS OF THE MYOTOME SOME CRANIAL NERVES HAVE SENSORY AFFERENTS I(OLFACTION), II(VISION), V(TOUCH), VII, IX, X (TASTE), VIII (HEARING AND BALANCE)

The bulges of the sensory ganglia of cranial nerves innervating the branchial arches are visible on the surface Arch 1: maxillary Surface bulge of sensory ganglion of Cranial nerve V (trigeminal) Arch 1: mandibular Surface bulge of sensory ganglion of Cranial nerve VII (facial) Arch 2 Mouse, 10 days, lateral view http://www.med.unc.edu/embryo_images/

Cranial neural crest cells give rise to structural components normally associated with the paraxial mesoderm in the trunk SEGMENTATION IN THE HEAD CRANIAL NEURAL CREST CELLS 1. SENSORY AND AUTONOMIC NERVE GANGLIA DERIVATIVES IN COMMON WITH 2. SCHWANN CELLS OF PERIPHERAL TRUNK NEURAL CREST NERVES 3. MELANOCYTES 1.BONE, DERMIS OF FACE 2. MENINGES OF BRAIN UNIQUE DERIVATIVES 3. CORNEA OF EYE 4. DENTAL PAPILLAE 5. CONNECTIVE TISSUE COMPONENTS OF BRANCHIAL ARCHES

In the facial region, neural crest cells contribute all of the skeletal and connective tissues with the exception of tooth enamel Arrows indicate the origin and destinations of neural crest cell populations. http://www.med.unc.edu/embryo_images/

The branchial arches contribute to features of the face with their tissue components deriving from both neural crest and myotome FEATURES OF THE FACE AND THEIR ORIGINS - 1 1. Unusually, supporting tissue components of branchial arches and face derive from neural crest EYE 2. Muscle contribution is from somitomeres (for example somitomere 4 gives rise to muscles of mastication) NASAL PIT 3. Maxillary arch extends inwards to fuse with its bilateral partner and the nasal structures. It forms the bone of the upper jaw and the tissues of the upper lip MAXILLARY DEVELOPMENT (from arch 1) STOMODEUM (mouth) 4. Mandibular arches fuse to form lower jaw TONGUE 5. Failure of fusion of maxillary arches and nasal prominences gives rise to cleft lip and palate MANDIBULAR ARCH(arch 1) (mastication) HYOID ARCH (II) (facial expression)

The epithelium of the oral cavity derives from both ectodermal and endodermal sources FEATURES OF THE FACE AND THEIR ORIGINS - 2 TONGUE NASAL CAVITY SECONDARY PALATE ORAL CAVITY TRACHEA OESOPHAGUS NASAL PIT MANDIBULAR ARCH LUNG BUD 1. Lateral walls of nasal cavity contain olfactory epithelium 2. the rest of nasal cavity is pseudostratified ciliated epithelium derived from the original ectoderm 3. The oral cavity develops partly from ectoderm and partly from endoderm. The fusion point between the two was the position of the (now degraded) oral plate

THE OTIC SENSORY PLACODES - 1 The otic placode is induced ectoderm which invaginates to become the cavity of the inner ear THE OTIC SENSORY PLACODES - 1 II VIII 1. The otic placode invaginates to form the otic vesicle which will become the inner ear 2. The splanchnopleure of the pharynx forms a diverticulum - the first pharyngeal pouch NEURAL TUBE (HINDBRAIN) OTIC PLACODE NEURAL GROOVE NOTOCHORD PHARYNX

THE OTIC SENSORY PLACODES - 2 Components of the middle and outer ear derive from the first pharyngeal pouch and first branchial cleft THE OTIC SENSORY PLACODES - 2 GANGLION OF CRANIAL NERVE VIII OTIC VESICLE / INNER EAR (from otic placode) BONES OF MIDDLE EAR (from 1st pharyngeal pouch) EXTERNAL EAR (from 1st branchial cleft) AUDITORY TUBE (from 1st pharyngeal pouch) Fish have just the inner ear as an organ of balance. The middle and outer ear evolved to receive and transmit sound waves

(A) (B) 9 day mouse 10 day mouse 9 day mouse http://www.med.unc.edu/embryo_images/ The neural tube in the region of the hindbrain induces formation of the otic placode (A) and then otic vesicle (B), dorsolateral to the pharynx The otic placode invaginates to form an otic pit and finally the otic vesicle. Its surface aspect is dorsal to the 2nd branchial cleft

THE DEVELOPMENT OF THE EYE - 1 The lens placode is induced ectoderm under the influence of the neuroepithelium of the optic cup THE DEVELOPMENT OF THE EYE - 1 ROSTRAL NEUROPORE LENS PLACODE FORE-BRAIN LENS VESICLE INNER/OUTER LAYERS OF OPTIC CUP (this neuroepithelial layer gives rise to the visual retina) OPTIC STALK

T H E D E V E L O P M E N T O F T H E E Y E - 2 The neuroepithelium gives rise to the pigmented and neural retinal layers of the visual retina T H E D E V E L O P M E N T O F T H E E Y E - 2 P R E S U M P T I V E C O R N E A I R I S P I G M E N T E D R E T I N A L L A Y E R N E U R A L L E N S R E T I N A L L A Y E R T E M P O R A R Y F U S I O N O F E Y E L I D S F I B R E S O F O P T I C N E R V E D E V E L O P I N G E Y E L I D

Mouse 8.5 days http://www.med.unc.edu/embryo_images/ Neuroectoderm of optic vesicle inducing surface ectoderm to form lens placode The invaginating lens placode pinches off to form the lens and invagination of the optic vesicle forms the optic cup connected to the brain via the optic stalk Mouse 11 days Mouse 10 days

REFERENCES Carlson BM (2003) Patten's Foundations of Embryology Noden DM, de Lahunta (1985) A Embryology of domestic animals McGeady TA, Quinn PJ, Fitzpatrick ES, Ryan MT (2006) Veterinary embryology University of North Carolina web site: http://www.med.unc.edu/embryo_images/