Harvey Lodish • Arnold Berk • Paul Matsudaira •

Chris A. Kaiser • Monty Krieger • Matthew P. Scott •

Lawrence Zipursky • James Darnell

Molecular Cell Biology

Fifth Edition

Chapter 15:
Integrating Signals with Gene Controls

    Copyright © 2004 by W. H. Freeman & Company

 Cell receives external signals

signal

receptor

Inducer Responder
(competence: past history)

   
Signaling systems and cell responses

   
   
   
   
   
   
 Experimental approaches

3. Genomic comparisons
conservation
diversity 

2. Detecting changes
mRNA
protein (level, modification, subcellular localization, 
interaction with others)
  
termporal changes (kinetics)

   
 Evolutionary conservation of core processes 
(~1500 genes common to human, fly and nematode)

~ 1/3 of genes are unique to human, fly and nematode
   
 Whole mount  Section Whole mount
(mouse embryo) (mouse embryo)  (fly embryo)

In situ hybridization
• Steady state RNA level of specific gene
Immunohistochemistry (Antibody staining)
• protein distribution

• Spatial and temporal pattern 
• Double, triple labeling
  • Specificity (cross­reactivity)
 
DNA microarray cluster analysis of gene expression

Microarray: DNA
Probe: mRNA (cDNA)

    Comparison: A vs. B
Protein microarray
• Protein­protein interaction
• Protein­lipid interaction

   
 Responses of cells to environmental influences
glucose/oxygen
Integrated regulation of glycogenolysis

dual inputs
cross­talk
coordination

   
 Secretion of insulin in response to a rise in blood glucose
Fasting: 5 mM
After meal  ↑

Km 20 mM

Muscle cells
Fat cells
(insulin receptor)

β cells => insulin => reduce blood glucose
α cells => glucagon => increase blood glucose

Antagonism => maintain homeostasis
   
 Insulin stimulation induces translocation of GLUT4­GFP 
from intracellular vesicles to plasma membrane

High blood glucose => pancreatic β islet cells 
=> Insulin => Fat cell => GLUT4 translocation to cell surface 
⇒ more glucose influx => reduce blood glucose

No insulin => surface GLUT4 internalize by endocytosis 
⇒less glucose influx

  Negative feedback loop => maintain homeostasis
 
 Responses to hypoxia
Sense [O2]

Rapid responses: protein activity
Slow responses: gene expression
Hypoxia­induced factor 1
(transcription factor)

   
O2 sensor 

   
Control of cell fates by graded amounts of regulators
Sequential induction of cell types

En Hh Dpp

Concentration gradient
thresholds
   
   
 Dominant marker (Myc)

    Loss of marker
   
 Flp­out clones

   
 Clonal mis-expression of upd
non-autonomously induced cell proliferation

lacZ (Upd)
BrdU

hs-FLP; Ay-GAL4, UAS-lacZ X UAS-upd

   
Tsai and Sun (2004) Genesis 39:141­153. 
 Inductive signaling

   
 Anchor cell

LIN­3
Vulval precursor cells LET­23

­ ­ LIN­3 ?
No AC or lin­3  or let­23  => all 3 o

­
let­23  in P5.p and P7.p  => 2o  LET­23 LIN­12
Suggest a relay signal from 1o (requiring let­23 in P6.p) 
MAPK (in P5.p, P7.p)
   
Development of a Drosophila oocyte into a mature egg

   
   
   
   
   
Setting up the polarity (body axes) of embryo

A-P, D-V axes already specified in oocyte (egg)

=> Determined by maternal genes
   
Dorsoventral axis determination
Reciprocal signaling between oocyte and follicle cells Nuclear translocation 
of Dorsal

Toll

proteolytic 
cleavages

   
Nuclear gradient of Dorsal protein define D-V axis

Dorsal Dorsal protein

Ventral WT dorsalized ventralized

Mutants Mutants
(in cytoplasm) (in nucleus)
   
 D­V gradient of Dorsal 
induces different target genes
along the D­V axis

   
repression

activation

   
 A-P and D-V axes specification during oogenesis

   
Dividing embryo into smaller units (segmentation)

embryo

larva

   
Gilbert, 2000, Developmental Biology, 6ed
   
Gilbert, 2000, Developmental Biology, 6ed
 WT

bicoid
mutant

embryo from bicoid mutant mother

   
Figure 8.11

   
A­P axis determination
Maternal bicoid mRNA localized to anterior of early embryo

After fertilization

   
 RNA localization
• Cis-element in RNA (untranslated region)
• Binding proteins
• Link to cytoskeleton

Cytoskeleton
(transport, anchorage)

5‘UT Coding region 3‘UT

   
 Localization element in bicoid mRNA 3’UT
   
 Transcription of hb is sensitive to Bicoid dosage

   
Bicoid activate different genes at different concentration

野生型 bicoid 突變

   
 Precise location of the posterior Hb boundary
independent of variation in [Bicoid]
independent of Gap genes

   
Patel & Lall, 2002, Nature 415:748-9
 Posteriorly localized

   
   
 Screening for zygotic-effect
embryonic lethal mutations
affecting segmentation

Eric Wieschaus &

Christiane Nusslein-Volhard
1995 Nobel Prize

   
Gilbert, 2000, Developmental Biology, 6ed
   
Gilbert, 2000, Developmental Biology, 6ed
 Pair­rule genes

   
 Pair-rule gene expression: seven stripes
even-skipped (eve)

   
Gilbert, 2000, Developmental Biology, 6ed
   
 The first three hours of embryo development

Syncytial Cellular
blastoderm blastoderm

Before gastrulation:
• No cell movement
• Single layer of cells: 2D
   
Gilbert, 2000, Developmental Biology, 6ed
 Complex cross-regulations

   
 Syncytial blastoderm
• No barrier between cells
• Free diffusion
• Nuclear factors

Cellular blastoderm
• No free diffusion
    • Signal through cell membrane
   
 Different Hox gene expression pattern
in different segments

   
 Segmental specificity: homeotic genes

bithorax ： T3 transform into T2

   
 WT Antennapedia
antenna  leg
   
   
Gilbert, 2000, Developmental Biology, 6ed
 HOX genes specify segmental identities

fly

mouse, human

   
Gilbert, 2000, Developmental Biology, 6ed
   
Gilbert, 2000, Developmental Biology, 6ed
 Hox genes
Homeotic genes
Homeo box
Homeo domain

Selector genes

Target specificity => cofactors

Isoforms
Target genes (realisator genes)
Master vs. micromanager?
Colinearity
Regulation of expression

   
 Floral organ identity
Arabidopsis thaliana

WT Flower­to­leaf 
homeotic transformation

   
   
 ABC model of floral organ identity

   
 Regulation of segment polarity genes expression

   
Gilbert, 2000, Developmental Biology, 6ed
 Creation of boundary between parasegments

Sharpening and maintenance of boundary

   
 Processing of Hedgehog precursor protein

   
 Hh signaling pathway

   
 Wnt signaling pathway

   
Graded induction of different cell types in neural tube
By Sonic hedgehog (Shh) and BMP signals

   
 Membrane­bound ligands

   
Reciprocal signaling (induction) during angiogenesis

   
 Amplification of an initial bias 
by Notch­mediated Lateral inhibition 

   
 Equivalent cells

   
   
 Notch activation

Delta Delta

   
 Competence

   
 Same signal =>  Diverse responses

e.g.  RTK­Ras­MAPK signaling pathway

1. Differences in strength or duration of signal
2. Differences in downstream pathway (competence; past history)
3. Combination with other signals

   
 Sequential signaling

RTK

Muscle founder cell
eve activated by combination of Dpp/Wg/RTK and muscle­specific factors

   
 Limb development depends on 
integration of multiple signal gradients

   
Signals are buffered by intracellular and extracellular antagonists

  Antagonist of antagonist