Cell autonomous fate specification

1
Lecture 14

• Cell autonomous fate specification
• cell division and growth

2
neuronal determination in flies

• formation of groups of neurons support cells
• in embryonic CNS
• in PNS sensory bristles
• made by group of 4 cells, from single precursor
  (sensory organ precursor, SOP)
• cell fates correlate with ancestry within group

Fig 11.5
Fig 11.9
3
Neuroblasts are modified epithelial cells

• in embryonic CNS, neuroblast is stem cell,
  generates ganglion mother cells
• in PNS, SOP divides within plane of epithelium

Fig 11.8
4
The sensory bristle lineage

• 4 cells, 4 fates
• are fates determined by ancestry or relative
  position in group?
• IIa non-neuronal
• IIb neuronal
• genetics look for mutants with no bristles or
  too many

5
numb mutants

• all cells socket, no neurons
• numb protein localized within SOP
• segregated to IIb daughter

wild type numb loss of function
6
Notch mutants

• opposite transformation to numb
• encodes cell surface receptor (numb may inhibit
  Notch function)
• implies cell-cell interactions within the 4-cell
  group

7
How does numb become asymmetrically localized?
apical

• epithelium has apical-basal polarity
• cells divide parallel to epithelium
• neuroblast divides along apical-basal axis
• numb localizes basally and so is segregated to
  one daughter

basal
SOP does not, but divides asymmetrically within
epithelium
8
Bazooka is required apically for numb localization

• Bazooka is the fly equivalent of PAR-3
• regulation of spindle orientation and cortical
  domains

epithelial
neuroblast
no bazooka numb everywhere random division
wild type
9
conservation of cell polarity machinery

• Worm embryo
• PAR-3/6 anterior, PAR-1/2 posterior
• Fly neuroblast
• PAR-3/Bazooka apical
• Vertebrate epithelia
• PAR-3/6 complex subapical
• PAR-1 basolateral

10
expression of PAR-4/LKB1 is sufficient to
polarize single cells

• mutant epithelial cell line, cannot form
  junctional complexes
• expression of LKB1 sufficient to form asymmetric
  brush border, etc
• cell-cell junctions not required...

Baas et al, Cell, Vol 116, 457-466, 6 February
2004 Complete Polarization of Single Intestinal
Epithelial Cells upon Activation of LKB1 by STRAD
11
Summary of cell asymmetry

• cell-autonomous mechanisms of determination are
  widespread
• conserved pathays that generate or maintain
  cellular asymmetry
• PAR proteins define non-overlapping cortical
  domains by inclusion and exclusion
• PAR domains regulate overall cell polarity
  (microtubule cytoskeleton?)
• cortex influences orientation of spindle and vice
  versa

12
Cleavage divisions

• early divisions of zygote, generate blastula
  (hollow ball of cells)
• divisions can be determinative (worms, ascidians,
  frogs)
• how are they organized in space and time?

13
Cleavage divisions special aspects

• No increase in mass (no growth)
• rapid cell cycle alternating S and M phases.
  G1, G2 (gap) phases arise later.
• maternally controlled. onset of zygotic
  transcription during cleavage.
• mammals are exception--slow, zygotic control

14
Three kinds of cleavage pattern
Fig 8.4

• differ in orientation of divisions and whether
  they are equal/unequal

15
Radial cleavage

• echinoderms, (ascidians, frogs)
• first two cleavage planes meridional, at 90 to
  one another
• third cleavage equatorial, creating animal and
  vegetal tiers at 8-cell stage

An
Vg
mitotic spindles
16
Radial cleavage

• Sand dollar Dendraster

17
Unequal cleavage

• C. elegans
• first division unequal
• division of AB are orthogonal division of
  zygote-- rotational cleavage (also in mammals)

18
Spiral cleavage

• Annelids, molluscs
• similar to radial except that spindles are tilted
  in spiral round An-Vg axis
• spiral has handedness (chirality)

Snail Limacina
19
Determinants of cleavage patterns

• Yolk--blocks movement of furrow
• relative positions, orientation of nuclei and
  centrosomes (asters)
• so, study in animals with little yolk

20
cytokinesis
astral microtubules
chromosomes
spindle microtubules
nuclear envelope breakdown
actin-myosin contractile ring (cleavage furrow)
21
What controls where the cleavage furrow forms?
ectopic Rappaport furrow
Fig 8.5

• Raymond Rappaport 1974, working on sand dollar
• conclusion position of centrosomes/asters is
  critical, spindle is not?

22
bipolar spindle not required, but central spindle
MTs may be special
see Canman et al 2003

• treat cells with drug (monastrol) that prevents
  centrosome separation
• form monopolar half spindles
• furrow still forms near chromosomal (not astral)
  spindle MTs

23
rappaport vs. canman

• both show furrow formation without bipolar
  spindle, but difference is whether astral or
  spindle MTs closest to furrow
• redundant pathways--if no spindle MTs, astral MTs
  can work?

24
C. elegans

• posterior movement of zygote nucleus does not
  happen in par mutants
• conclusion cortical domains can affect position
  of nucleus/centrosomes/MTs within the cell
• signaling pathways still being worked out

WT
par
25
orientation of centrosome pair
centrosomes separate
duplicate and migrate
rotate only in P1
Fig 8.6
why do centrosomes rotate in P1?
result AB divides at 90 to previous axis P1
parallel
26
cortical attachment sites re-orient the
centrosomes in P1

• Hyman White 1990
• are MTs from asters attracted to remnant of
  previous cytokinesis?
• experiment cut MTs from one centrosome (with
  laser) other centrosome moves towards remnant
  site
• cortical sites can control centrosome
  orientation cytokinesis dependent

27
PAR proteins affect centrosome orientation in
later cells
rotates
Wild type
par-3

• model PAR-3 inhibits rotation in AB
• PAR-3 overrides effect of cytokinesis remnant?

par-2 Both rotate
par-3 par-2 Looks like par-3 par-3 is epistatic
to par-2
28
maternal control of cleavage pattern

• Crampton, 1894, in Lymnaea
• variants with left- and right-handed shells
• early cleavages are also mirror-image!
• recessive maternal-effect mutation (d)
• can be rescued by injection of cytoplasm from
  wild-type

wild type dextral spiral
d/d mutant sinistral spiral
29
a cleavage clock in echinoderms
M
M
E
shaking in dilute buffer
M
E
shaking in dilute buffer
M
E

• Sven Hörstadius, 1973
• conclusion timing independent of cytokinesis

30
Summary

• orientation and position of nucleus/centrosomes
  controlled by maternally supplied components of
  cytoplasm (cortex)
• cytokinesis-dependent mechanisms (remnants of
  cleavage furrows)
• cytokinesis-independent mechanisms (cleavage
  clock)

31
control of cell division and growth

• early cleavage divisions are rapid because embryo
  does not grow
• later coordination of cell division and growth
  of organism/organ

32
early cleavage divisions

• Cleavage cell cycles are short and synchronous
• Xenopus 35 minutes
• C. elegans 20 minutes
• Drosophila 8 minute nuclear division cycle
• exception mammals--24 h

33
the mid blastula transition (MBT)

• Xenopus
• cycles 1-12 are rapid and synchronous
• then cell cycle elongates, G phases added
• lose synchrony
• cells become motile
• activation of zygotic transcription
• what determines timing of MBT?
• cytochalasin blocks cytokinesis (but not DNA
  replication)--no effect

Fig 3.32
34
The nucleocytoplasmic ratio

• in cleavage, DNA content rises exponentially,
  total volume of cytoplasm is constant
• nucleocytoplasmic ratio is increasing
• Newport Kirschner 1982 inject DNA into frog
  egg, MBT starts early--embryo thinks it already
  replicated DNA
• (does not involve specific DNA sequence)

35
MBT in Drosophila

• cycles 1-10 rapid
• transcription starts cycle 11, gradual addition
  of G2 phase in cycles 11-14
• cycle 14 is asynchronous synchrony within
  mitotic domains

Fig 14.3 the mitotic domains
36
nucleocytoplasmic ratio in fly

• haploid embryos undergo 1 extra cycle before MBT
• confine nuclear divisions to one half of
  egg--this half will undergo premature MBT
• model nuclear factor (DNA or protein?) titrates
  a cytoplasmic factor
• when concentration of cytoplasmic factor drops
  below threshold level, MBT starts?

37
rate-limiting step in cell cycle

• Cell cycle engine cyclin-CDK complex
• CDKs drive mitosis activated by removal of
  inhibitory phosphates
• phosphatase called string (cdc25)
• early embryos components in excess, cell cycle
  is free-running
• after cycle 13 maternal string runs out CDKs
  inhibited until zygotic string made
• mitotic domains--differences in rate at which
  string made

38
how is the N/C ratio read?

• Simple idea it is not read just there is no
  time for transcription until cells run out of
  maternal protein, allowing G phases?
• Evidence that specific factors required to read
  N/C ratio comes from Drosophila genetics
• mutants that fail to pause cell cycle in cycle 14
• tribbles, fruhstart
• normal function is to pause cell cycle (i.e.
  opposite from string) presumably in response to
  N/C ratio
• but do not seem to inhibit string directly
• later development--cell division rates controlled
  by limiting amounts of growth factors

39
growth control

• how are sizes of tissues, organs, animals
  controlled?
• growth increase in mass
• cell proliferation
• cell size increase
• increase in ECM
• involves extrinsic signals (e.g. growth hormone,
  nutrition) and intrinsic programs

40
cell size

• function of DNA content (ploidy) in most organisms

e.g. plants (Datura)--cells can get bigger if
they have more DNA (endoreplication)
cleavage division without growth endoreplication
growth without division
41
in absence of endoreplication, growth factors may
be limiting

• excess nutrients do not cause cells to get bigger
• excess nutrients growth factors and do

fly cells (clones) overexpressing insulin
receptor or Myc cells are bigger than neighbors
(hypertrophic)
42
growth rate controlled by intrinsic programs

• graft limb bud from large species to flank of
  small species

Figure 14.5
43
organ size does not depend on cell number
cross sections of renal ducts, all to same scale

• Gerhard Fankhauser, 1945
• newts with different ploidy are same size
• organs, tissues all compensate

44
competition mechanisms must regulate organ size
/ cells (yellow) in Minute/ background
proliferate faster to make a large clone...but
wing is normal size!
Fig 5.26