Lab 8: Chick 72 hoursLab 8: Chick, 72 hours

Nervous systemCranial nervesCranial nerves

Digestive system

72 hour chick embryo

• Cranial nerves and ganglia are quicklyCranial nerves and ganglia are quickly developing, with one new cranial ganglion per neuromere.p

• The organs of the digestive system areThe organs of the digestive system are forming.

• At this stage the embryo has a foregut, hindgut, and the beginnings of organshindgut, and the beginnings of organs along the midgut.

Neural crest cells• So versatile that they could be called a

fourth germ layer. They help form many titissues:– Melanocytes, adrenal glands, peripheral

system neurons including spinal ganglia, andsystem neurons including spinal ganglia, and bones and cartilage of the head, and parts of the cranial trigeminal and glossopharyngeal and vagus gangliaand vagus ganglia...

• They arise along the neural tube startingThey arise along the neural tube starting around 27 hours and are migratory along specific paths. Two movies show the fluorescently labeled cells moving awayfluorescently labeled cells moving away from the neural tube over 10-12 hrs.

Neural crest cells migrating

Neural crest cells migrating

Neural crest cells can be fluorescentlyNeural crest cells can be fluorescently labeled with a specific antibody (HNK-1)

Immunocytochemistry for neural crest cells

HNK 1HNK-1 expressing cells in color

Neural crest cell derivativesNeural crest cell derivatives

Origin between ectoderm and neural tube

• Neural crest cells play a large role in vertebrate evolution because they form sovertebrate evolution because they form so many of the structures in the head and pharyngeal arches that are characteristicpharyngeal arches that are characteristic of a vertebrate body type.

• They are therefore the main cells responsible for changes in craniofacial evolution.

The avian bill is formed in part from the cranial neural crest migrating from the fore and midbrain

i t th b hi l hregion to the branchial arches

(A): Migration of neural crest cells (shown in red)

(B) and (C): Maxillary (mx) and mandibular arches(B) and (C): Maxillary (mx) and mandibular arches (md)

(D): Beak bone (red) from NCC(D): Beak bone (red) from NCC.Trainor, P. ( Science 299: 523 2003).

The avian bill is not entirely made from NCCs – they contribute but do they determine its pattern?contribute but do they determine its pattern?

A grafting experiment wasexperiment was done to test this. Chick neural crest The duck beak

of course iscells were put in the quail embryo (“quck”) or quail

of course is longer. The quail beak has

ill( quck ) or quail neural crest cells were put in the duck embryo (“duail”) The

a maxillary bump near the tip (egg tooth).embryo ( duail ). The

results are on the next slide.

p ( gg )

A. Quail NC cells B. Duck NC cells cells transplanted in a duck embryo ( k) lt

transplanted in a quail embryo (a duail) results in a (a quck) results

in a stubby quail type bill and

)longer duck type bill.

having the egg tooth bump.

• The results of the reciprocal transplants indicate that it is the neural crest whichindicate that it is the neural crest which forms not only the cartilage and bone but directs the overlying ectoderm and thedirects the overlying ectoderm and the mesoderm as well in the formation of the avian beakavian beak.

Cranial nerves and gangliaSensory organsSensory organs

• Most of the paired cranial nerves (bundled collections of neurons) have a cell bodycollections of neurons) have a cell body containing the nucleus and most of the cytoplasm in ganglia (singular ganglion). The thi d f th d i th h lithird, fourth and sixth have no ganglia.

• Nerves are made up of neurons extending back• Nerves are made up of neurons extending back to the central nervous system or to some target sensory organ or muscle.

• The next slide shows a typical migrating embryonic neuron with a leading edge growthembryonic neuron with a leading edge growth cone which extends the axon from the cell body.

The migrating neuron as shown in lecture.

Cranial nerves• The sources of the neurons can be alongside

the neuromeres (rhombomeres), or from ectoderm just under the epidermis (epibranchialectoderm just under the epidermis (epibranchial placodes) or growth from the developing sensory organ (nose, eye, ear).

• The olfactory sensory nerve (I) forms axons growing from the nasal pits toward the cerebral hemispheres.hemispheres.

• The optic nerve (II) forms from the sensory retina and later axons grow back to join to the diencephalondiencephalon.

• The oculomotor (III) nerves are motor and later innervate the eye muscles (not yet formed).y ( y )

Cranial nerves of the hindbrain

• The trigeminal (V) is both sensory and motor with the semilunar ganglion sending branches to the eye (ophthalmic) and maxillary/mandibular processes. Placode cells and neural crest form pthis nerve.

• The facial nerve (VII with acousticofacialis ganglion) is a mixed nerve for the face Trace itganglion) is a mixed nerve for the face. Trace it to the second mainly, but mandibular arch too. Placode cells and neural crest contribute to this nerve It is close to the auditory nerve (VIII)nerve. It is close to the auditory nerve (VIII) which innervates the nearby developing ear (auditory vesicle).

• The glossopharyngeal (IX) and associated superior ganglion is a sensory nerve cansuperior ganglion is a sensory nerve can be traced to the third arch. Epibranchial placodes and neural crest form this nerveplacodes and neural crest form this nerve.

• The vagus (X) and associated jugular ganglion is a mixed nerve for the fourthganglion is a mixed nerve for the fourth arch. Epibranchial placodes contribute to the neural crest forming these neuronsthe neural crest forming these neurons.

GlossopharyngealSpinal

Spinal cord

Glossopharyngealganglion (IX)

Otic vesicle

paccessory nerve (XI)

Otic vesicle

Acousticofacialis ganglia (VII/VIII)

left right Neuromeres ofmyelencephalon

Metencephalon

Yolk sacSemilunar ganglion of

MesencephalonYolk sac g g

trigeminal nerve (V)

anterior

Spinal cord

Notochord

Facial nerve

Semilunar ganglion (V)ganglion (V)of trigeminalnerve

Isthmus

Metencephalon

IsthmusMesencephalon

Digestive system and endoderm derivatives

• At 72 hours the endodermal derivatives such as the lung buds begin to rapidly expand into thethe lung buds begin to rapidly expand into the mesenchyme of mesoderm origin.

• The esophagus, stomach, liver and duodenum p g , ,are just beginning to form. Try to trace the connections between these tissues as they all branch off the same original endodermal tube.

• Note the closeness of the ductus venosus to the li di t l t th li ill d thiliver rudiment; later the liver will surround this vessel. Why would later much more vitelline blood be directed to the liver?blood be directed to the liver?

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Pharyngeal region• The four pharyngeal (branchial) arches are

characteristically found in all vertebratescharacteristically found in all vertebrates during development whether or not gills are formed. This is an example of the retention of common structures and developmental pathways in vertebrate

l tievolution.• The derivatives of the endoderm and

l t ll th t ineural crest cells vary the most in evolution.

Posterior endoderm derivatives• The hindgut is the region posterior to the

caudal intestinal portal The hindgut endscaudal intestinal portal. The hindgut ends in the cloaca. Note how the allantois branches off ventrally The cloaca is abranches off ventrally. The cloaca is a blind ending but will later form an opening at the site of the cloacal membraneat the site of the cloacal membrane (proctodeum). Try to find possible functional openings of the mesonephricfunctional openings of the mesonephric ducts into the cloaca.

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Sagittal section to show how allantois gbranches off of the hindgut

Neural tube cell organization• The dividing population of cells is closest to the

lumen in the ependymal (ventricular) layer as mentioned in the lecture. At the early stages seen in the next slide the division into three layers (ependymal mantle and marginal) is notlayers (ependymal, mantle and marginal) is not completed.

• Because all tube cells are attached at the ependymal end and extend the full width, the tube is considered to be a pseudostratified epithelium Pseudo meaning that there is onlyepithelium. Pseudo meaning that there is only one cell layer but because cell bodies are at various positions it looks like there are many ystrata or layers.

Chick embryo neural tube cells before the formation of the three neural layers.

Dividing cells can sometimes be seen close to the lumen in the ventricular zone.

anaphase

• In the spinal cord or brain of the 72 hour chick embryo the thin outer marginal zone sometimes is seen (i.e rarely) as distinct from the two darker and denser inner layers. Try to find it.and denser inner layers. Try to find it.

In viewing sections note how the thi k f th l i b tthickness of the layers varies between brain regions.

• In the next slide the adult spinal cord structure is shown with the dorsal sensorystructure is shown with the dorsal sensory and ventral motor nerves.In the 72 hr chick the nerve axons are• In the 72 hr chick the nerve axons are growing toward the targets (such as the limb bud which is very early in itslimb bud which is very early in its development) but the staining method used for these sections does not allowused for these sections does not allow nerve fibers to be easily seen.

Adult neural tube structure

• The oculomotor nerves (cranial nerve III) can sometimes be seen as thin usuallycan sometimes be seen as thin usually non-continuous streaks extending from the mesencephalon floor toward the internalmesencephalon floor toward the internal carotid arteries. These nerves will innervate the muscles of the eyes (Theinnervate the muscles of the eyes. (The oculomotor nerves may not be seen in all sets of sectionssets of sections.

Body cavities• The coelom is beginning to become

divided into the pericardial cavitydivided into the pericardial cavity surrounding the heart, the pleural cavity surrounding the lungs and the peritonealsurrounding the lungs, and the peritoneal cavity around the gut.