Molecular developmental biology III'

1
Molecular developmental biology III.

• Cell movements and the shaping of the vertebrate
  body

2
The polarity of the vertebrate (Xenopus) embrio

• The polarity of the embrio is based on the
  polarity of the egg animal and vegetal pole
• Vegetal pole (gut, internal tissues) VegT
  protein and the bound mRNAs, Wnt signal proteins
  and the Dsh proteins
• The sperm entry at the animal pole induces
  rotation of the actin rich egg cortex
• The animalis pole is rotated with about 30o to
  ventral direction, the VegT, Vg1 and the Dsh are
  separated, the Dsh determines the dorsal side
• The movement of the cortex depends on the site of
  the spermium entry and may have connection with
  the sperm centrosome

3
The blastula (Xenopus, vertebrate)

• Formed by serial cell divisions, filled inside
  with yolk
• After 12 cycles the cells begin to show the
  interphase (G1-S-G2), earlier they do not
• Cells giving rise to ecto-,endo- and mesoderm are
  distinct
• The cells are connected with (tight and gap
  junctions)
• The vegetal cells with molecules of TGF?
  superfamilly are influencing the mesoderm forming
  cells above them
• The dorsal blastomer modify Wnt signals, this
  forms the dorsal lip indicating the beginning of
  gastrulation

4
The gastrulation

• Ordered cell movements creating three germ layers
• It starts at the dorsal side - determined by the
  cortical movements - with the formation of the
  blastopore at the vegetal pole
• The upper lip of the dorsal blastopore has a
  special role, it is called organizer
  transplanting this part to an other blastoderm
  results a double embryo (Siamise twins) in frog

5
The convergent extension is the start of
gastrulation

• Cells around the organizer in the dorsal midline
  will extend into diagonal direction
• The basal part of the bottle shaped cells will
  narrowe and sink toward the center of the
  blastoderm
• The neighbouring cells will clime on them and
  push them downwards.
• The Wnt-binding receptor Frizzled regulates
  Dischevelled which plays a role in the whole
  process

6
Gastrulation

• The mesoderm is created continuously at the
  surface of the ectoderm and mesoderm during
  gastrulation
• The mesoderm cells are migrated on fibronectin
  layer
• Ca2 dependent cadherines play a role not only in
  gluing cells together but transfer signals and
  connect to actin inside the cells
• The cells after isolation and separation may
  reagregate together again into a nearly
  blastodermal stage

7
Neural tube and notochord formation

• after gastrulation the mesoderm is divided into
  three parts notochord (from organizer) and two
  lateral mesoderms
• The notochord gradualy extends to posterior
  direction and sink ventrally
• Parallel with this the neural plate, the neural
  tube and the neural cells are formed

8
Somite formation

• From the lateral mesoderm
• Somite borders are marked by protocadherin
  expressing cells
• Controlled by c-hairy-1 (pair-rule homologe)
  oscillation pattern
• The c-hairy-1 is expressed in posterior part of
  presomital mesoderm in 90 minutes cycle, in the
  posterior part of the somite cell division is
  stopped at the peak of the cyle and the same
  thing occurs during the cycle in the anterior
  part
• Dermis, sclerotoma and myotoma are formed from
  the somites

9
Embryonal tissues are created after cell
migrations

• Enviromental signals are important, these signals
  are exchanged between migrating cells
• Migrating cells follow paches of embryonic
  connective tissue
• Coordinated by special transcriptional machinery,
  myoblasts forming limb muscle express myoD and
  those forming trunk muscle express myf-5
• Migrating cells might be blood cell precursors,
  germline cells, neurons of the central nerv.
  syst. endothel cells forming blood vessels

10
Migration of crista neuralis cells

• Create periferal neurons, glias, epidermal
  elements, pigment cells, (head connective tissue
  elements bones of skull and jaws, adrenal cells
  the fourth germ layer
• The endothelin-3 is important, secreted by the
  surrounding tissues, in lack of it unpigmented
  stripes on the skin and megacolon are formed
  because intestine lacks innervation (Hirschprung
  dis.)
• The germ line cells, pigment cells and neurons
  carry Kit receptor on their surface recognised
  by the steel ligand and this influence migration

11
The effect of mutation in the Kit gene

• The infant and the mouse are both heterozygous
  for the loss of function mutation of the Kit
  receptor gene (possess only one perfect copy of
  the gene), and this effects pigment cell
  formation in homolog regions

12
Formation of the left and right side of the body

• The 99.98 of people have their heart on the left
  side and the other internal organs (stomach,
  liver) oriented according to this (but in situs
  invertus)
• The formation of this pattern has two component
  1. the creation of asymmetry of the two half 2.
  the orientation of the two side
• In humans and mice a number mutation is known
  where the two sides are exchanged, these are
  traced back to the molecular asymmetry in the
  nodus (called organizer in chicken, mice, frog),
  50 of the mutations show normal body
  organization.

13
Molecular asymmetry in the node

• It expresses TGF? type proteins (i.e. Nodal, Shh)
  in an asymmetric way
• These protein regulates each other
• KO mutation of the Lefty-1 gene results in
  mirror-image like duplication
• the asymmetria is related to the circular
  movement of cilia which create a current to the
  left
• 50 of men showing fertility problem, cronic
  bronchitis and sinusitis together had situs
  invertus because of defect of dynein, and the
  effect on ciliary beating

14
The mouse as a developmental biological organism

• It has gained a special importance compared to
  Drosophila and the Nematoda because the results
  are easier to transfer into medical research
• Human proteins in 80-90 are identical with the
  mouse proteins
• Mouse embyos can be manipulated and studied
  without killing them
• The mouse egg is 2000 smaller than the frog egg

15
The starting development of the mammalian embryo

• The zygotic transcription is started already in
  the two cell stage
• Little yolk, nutratives are provided by the
  placenta, amniotic sac
• The zygote divides inside the zona pellucida, the
  blasztocyst are formed in the 16 cells stage,
  folded by the trophectoderm
• On the inner side of the multicellular layer of
  the trophectodrem the embryonic bud is formed
  (the embryo at this time already in the uterus)
• The blasztocyst cells are totipotent, can be used
  to create chimeric animals - the embryonal stem
  cells transplanted into kidneys or testis create
  teratoma (epidermal, bone, glandular epithel
  cells together), this is a form tumorous growth
• Embryonic bud then embryonic shield, germ layers
  are developed, neurulation begins

16

• Totipotent germ cells can be cultured and
  modified genetically
• knock-out mice are made in this way first the
  perfect gene is exchanged in cultured germ cells
  into a modified versiont, a cell line is
  established and these recombinant cells are
  transplanted into the blastocyst of a female
  mouse, with a little luck these cells give rise
  to the germ line and form oocytes then becomes
  fertilised and become founders of a new
  generation of mutants which sometimes can be made
  homozygous

17
Branching morphogenesis of the lung

• (A) the morphogenesis of bronchial structure is a
  good example of dichotomic branching (B) the
  bronchial tree of the lung

18
The three stages of neural development

• The first is not neural specific, the third also
  last in adult age
• Neural cells are the oldest specified cells
  1011 neurons in humans, each can connect to
  thousand others, but these are not so precise
  like the computer connections different
  neurons, glias, sensory cells and proprioceptors
  can be connected

19
The central nervous system is formed from the
neural tube, this is regulated by morphogens
(i.e. Hox genes), resulting in a dorsoventral
pattern.
20
Programed production of different type of neurons
Neuron progenitors divide close to one surface of
the cortical neuroepithelium and daugther cells
are crawling along the surfaces of radial glia
cells. The first-born neurons settle closest, the
later borned ones migrate further out.
21
Axonal growth, motoric synapse

• The axons develop with a growth cone, that is
  permanetly testing its enviroment and can grow 1
  mm dailly
• The N-cam and the Ca2 dependent cadherines are
  important in axonal growth
• The dentrite and the axon tarnsport different
  materials
• The spinal cord neural cells expressing Islet/Lim
  protein of the homeobox family develop into motor
  neurons and form connection with muscles
  expressing the same protein

22
Axonal growth in the developing spinal cord
The developing axonal growth cone (GC) is
attracted to the floor plate, because it
expresses netrin receptor (dcc) and the floor
plate produces netrint. When the GC reaches the
floor plate it produces a protein called
roundabout, which is a receptor for the repellent
slit produced by the floor plate and inhibits dcc
expression. At the same time an other receptor
is expressed in the GC for the repellent
semaphorin secreted by the side wall of the
neural tube. The axon is trapped and directed
toward the brain, reaching that it fights for NGF.
23
The axonal growth

• The axons reachig their target are guided by
  tissue growth factors neutrophin family, i.e.
  NGF directs neurits to smooth muscle cells, its
  receptor is a tirozin kinase, TrkA
• The neural cells are competing for NGF, that is
  necessary for their survival (50 of sensory and
  motor neuron will die before reaching target
  tissue)
• The death of neural cells follows internal
  program (apoptosis), cells otherwise looking as
  healthy die fast in lack of NGF, the whole
  process can be reverted by supplying NGF

24
The neural path from eye to brain in zebrafish
(A) Retinal ganglion cells and the optic tectum
in the vertebrate (except mammals) midbrain
axons from nasal retina connect to the posterior
tectum and from temporal retina to the anterior
tectum (B) Tracer dyes taken up by retina neurons
reveal retino-tectal connections
25
Selectivity of retinal axons

• The optic nerve of a frog regenerated after
  cutting (1940), if the eye was turned with 180o
  the animal see the world upside down
• In vitro culture optical from temporal retina
  grow only to the anterior (A), the nasali neurons
  only to the posterior tectum (P) extract
• Explanation 1. The tectum has an ephrin
  gradient growing in antero-posterior direction 2.
  The axons of the nasal neurons has little, while
  the axon of temporal neurons has a lot of ephrin
  receptor
• Az ephrinA is a GPI (gluco-phoszphoinositol)
  protein, binding to a receptor tyrosin kinase
• An other, not well known dorsoventral gradient
  also takes part in the selecting process

26
The homunculus

• Formed by a similar mechanism to the retinotopic
  map, a somatosensory map shows wich part of our
  body is mapped to a particular zone of the
  somatosensory cerebral cortex(homunculus)
• the acustic map is tonotopical, the sound from
  different pitch are ordered according to a linear
  order, similar to the keys of a piano

27
Renewal of synaptic relations (in retinotectal
connections)
28
Synaptic connections are formed in use
(A)Normally stripes are driven by the right eye
alternate with stripes, of equal width, of the
left eye. (B) Keeping one eye covered during the
critical period of development, its stripes
shrink and those of the active eye expand. After
a time in covered, the vision of one eye can be
lost almost entirely (in humans ending at age of
5 years).
29
Synapse remodeling and memory

• The molecular mechanism of developmental synapse
  remodeling and adult memory may be similar
• two neurones are stimulated by a quick
  consequtive way, this will open the chanels
  regulated by the NMDA glutamate receptors giving
  a way for the entry of Ca2 into the postsynaptic
  cells
• This Ca2 entry influences pre- and postsinaptic
  cells strengthening the synapsis between them
  this condition is called the Hebbs rule, has
  been suggested to be the fundamental principle of
  associative learning