Молекулярная генетика развития

Молекулярная генетика развития
Вельков В В 2013
Gene Regulation during Development

Morphogenesis - How do you get from a spherical egg to say a frog?

Dolly and Bonnie
Gilbert, SF (2003) Developmental Biology, 7th ed.
The nucleus from an differentiated adult cell (from the mammary gland) was able to replace the nucleus
of a fertilized egg and produce an adult complete with functional gametes: Dolly had a daughter – Bonnie.
SO: during development genes are just turned on and off in a carefully orchestrated fashion. SO, THE DOGMA OF DEV. BIOL. Is differential gene expression controls development

The Spemann Experiment
Spemann & Mangold, 1923

Spemann Experiment
Outline: Graft a tissue from one embryo into another embryo, and see what happens – Cut and Paste
Main observation: A graft of a specific tissue (the organizer) to a specific location can induce Siamese twins connected at the belly.
Conclusion: Hilde Mangold and Hans Spemann
found that a piece of
Prospective dorsal tissue
can organize formation
of an embryo

“A piece taken from the upper blastopore lip of a gastrulating amphibian embryo exerts an organizing effect on its environment ...
Such a piece can therefore be designated as an organizer”
Vegetal view

Spemann won the Nobel prize in 1935
Hilde Mangold died in 1926…
“Spemann Organizer” was found
in all vertebrates, including human
The organizer secretes both morphogens
and their inhibitors which diffuse
throughout the embryo

Universal mechanism of animal development
Gene expression controls 4 essential process
Short- long-range diffusible signaling molecule
Changes in cell adhesion

Development is progressive
Specification of cell fate: determination
All cells still ‘look the same’
Can be tested by transplantation experiments
Interactions can make cells different from each other: induction

In the process called induction
Signal molecules from embryonic cells cause transcriptional changes in nearby target cells
Early embryo
(32 cells)
NUCLEUS
Signal
transduction
pathway
Signal
receptor
Signal
molecule
(inducer)

From single cell to organism – a life cycle
The use of a model organism
Fertilisation followed by cell division
Pattern formation –
instructions for
Body plan (Axes: A-P, D-V)
Germ layers (ecto-, meso-, endoderm)
Cell movement - form – gastrulation
Cell differentiation
Cell growth, cell death (apoptosis)

Three Strategies:
mRNA localization
Cell-to-cell contact
Signaling through the diffusion of secreted signaling molecules

Морфогены
и рецепторы
морфогенов

Morphogen – substances that define different cell fates in a concentration-dependent manner
Interaction of two signaling centers located in the anterior and posterior poles of the egg pattern insect body axis

Morphogen = Soluble molecule that causes cellular commitment but is secreted some distance
from the target cells.
Morphogen Gradient = concentration gradient of a morphogen.

Градиенты морфогенов
Белки, кодируемые генами, функционирующими в ходе созревания яйца, и транспортируемые туда из питающих клеток,
распределяются по оси яйца,
образуя градиенты, специфически характерные для продуктов каждого гена

Morphogen Threshold Concentrations
embryo

Morphogen Threshold Concentrations
embryo
Morpho-
gen
conc.
position

Morphogen Threshold Concentrations
embryo
Morpho-
gen
conc.
position

Morphogen Receptor
Some mRNAs Become Localized within Eggs and Embryos due to an Intrinsic Polarity in the Cytoskeleton
The asymmetrically distributed mRNA is transported along element of the cytoskeleton
from – to the growing + end.

Adaptor protein
- The morphogene receptor
binds to 3’untranslated trailer
(3’UTR) region
of morphogene mRNA,
A morphogene receptor
has two domains:
one recognizes the 3’ UTR,
the other
associates with myosin.
Cytoskeletal elements –
microtubules are polarized within
the oocyte and can be used to allow
the localization of mRNA molecules
to specific parts of the cell.
morphogene
receptor

Influence of Other Cells
Morphogen Receptor Gradient = frequency gradient of the receptors for a morphogen in target cell cell membranes.
Morphogen gradient
Morphogen receptor gradient

Morphogens
Coordinate the cell growth and differentiation.
Formation of a long-range concentration profile.
Cellular response is concentration dependent.

Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Life cycle

Many Adult Structures Develop
from Imaginal Discs in Larvae and Pupae

Position or Spatial Organization is Everything
2 main mechanisms used to communicate positional information
Morphogens
Cell adhesion

Fate Map: all different regions of an embryo develop is described
Developmental program — process for cell achieving its fate

Морфогены
активируют
Гены сегментации
активируют
Гомеозисные гены
активируют…

Genetic Analysis of Early Development:
Scientific Inquiry
The study of developmental mutants
Laid the groundwork for understanding
the mechanisms of development

Drosophila Embryogenesis
First phase is establishment of body axes
Before fertilization, specialized nurse cells
move maternal mRNAs (morphogenes) into maturing oocyte
Then morphogens are distributed in the oocyte prior to fertilization
The Morphogens organize the structure of the egg
These positioned mRNA will initiate a cascade
of gene activations following fertilization
Яйцо созревает в особой камере — фолликуле.
В фолликулах - гены с материнским эффектом, которые функционируют
еще до оплодотворения яйца сперматозоидом
В фолликуле ооцит и 15 огромных питающих клеток,
которые синтезируют «РНК морфогены» и перекачивают их в ооцит.

At the start of development, gradients are established in the egg
along two axes, anterior-posterior and dorsal-ventral.
Anterior
(head)
Posterior
(tail)
(underneath)
(top)

Maternal gene products, called morphogens, establish gradients in early embryogenesis.
2. Anterior-posterior development uses localized gene regulators.
3. Dorsal-ventral development uses localized receptor-ligand interactions.

Градиент морфогенов
активирует
зиготные гены
После того как градиенты в яйце созданы, происходит оплодотворение и начинается дробление зародыша
образуется однослойная бластодерма. Каждая клетка в ней занимает определенное положение по отношению к сформировавшимся градиентам,
- обладает определенной позиционной информацией.
Морфогены взаимодействуют с регуляторными участками генов, активирующихся у зигот (зиготических генов).

Expressed during oogenesis by the mother. Act upon or within the maturing oocyte.
Expressed after fertilization. Mutations in these genes alter the number or polarity of segments. Three groups of segmentation genes act sequentially to define increasingly smaller regions of the embryo.
Control the identity of a segment, but do not affect the number, polarity or size of segments. Mutations in these genes cause one body part to develop the phenotype of another part.

Establishment of the A/P axis
Nurse cells secrete maternally produced bicoid and nanos mRNAs into the oocyte
-Differentially transported by microtubules to opposite poles of the oocyte
-bicoid mRNA to the future anterior pole
-nanos mRNA to the future posterior pole
-After fertilization, translation will create opposing gradients
of Bicoid and Nanos proteins

bicoid (bcd) Encodes the Anterior Morphogen
.

Bicoid gradient
Bicoid acts as a transcriptional activator of hunchback gene transcription

Nanos gradient

Establishment of the A/P axis
Bicoid and Nanos control translation of two
other maternal mRNAs, hunchback and
caudal, that encode transcription factors
-Hunchback activates anterior structures
-Caudal activates posterior structures
The two mRNAs are not evenly distributed
-Bicoid protein inhibits caudal mRNA translation
-Nanos protein inhibits hunchback mRNA translation

How Bcd Protein Works

Establishment of the D/V axis
Maternally produced dorsal mRNA
is placed into the oocyte
-Not asymmetrically localized
Oocyte nucleus synthesizes gurken mRNA
-Accumulates in a crescent on the future dorsal side of embryo
After fertilization, a series of steps results in selected transport of Dorsal into ventral nuclei, thus forming a D/V gradient

Segmentation genes act sequentially to divide the embryo into segments
Normal Drosophila embryo divided
into 15 segments
3 head, 3 thoracic and 9 abdominal
Each will give rise to unique morphological features in adult

Production of Body Plan
The body plan is produced by sequential activation of
three classes of segmentation genes
Segmentation Is Initiated by Localized RNAs
at the Anterior and Posterior Poles of the Unfertilized Egg
1. Gap genes
-Map out the coarsest subdivision along the A/P axis
-All 9 genes encode transcription factors that activate the next gene class
2. Pair-rule genes
-Divide the embryo into seven zones
-The 8 or more genes encode transcription factors
that regulate each other, and activate the next gene class
3. Segment polarity genes
-Finish defining the embryonic segments

Gap genes
Expressed first
Gap mutants show a gap in segmentation pattern at positions where particular gene is absent
Binding sites in promoter have different affinities for maternal transcription factors
Gap genes encode transcription factors that influence expression of other gap genes

Gap genes
Hunchback, krüppel, giant, tailless and knirps.
Their expression patterns in the early embryo
are determined by the maternal effect gene products
These genes establish the segmented body plan
of the embryo along the anterior-posterior axis.

The A-P axis is divided into broad regions
by gap gene expression
The first zygotic genes
Respond to maternally-derived instructions
Short-lived proteins,
gives bell-shaped distribution from source

Zones of Expression of Four Gap Genes:
Hunchback, Kruppel, Knirps, and Giant in Late Syncytial Blastoderm Embryos

Defects in Segmentation from Mutations in Gap Genes
A)
B)
C)
D)

Mutations in Gap Gene Result in Loss of Segments Corresponding to Zone of Expression

Transcription factors in cascade
GAP genes
Hunchback (hb) , a gap gene, responds to the dose of bicoid protein
A concentration above threshold of bicoid activates the expression of hb
The more bicoid transcripts, the further back hb expression goes

Krüppel reads two values
Krüppel (Kr), a gap gene, responds to the dose of hb protein
A concentration above minimum threshold of hb activates the expression of Kr
A concentration above maximum threshold of hb inactivates the expression of Kr
GAP genes

Homeotic mutations
We know some misterious mutations,
which generate horroristic monsters

Homeotic-Selector/ HOX Genes

Production of Body Plan
Segment identity arises from the action of homeotic genes
-Mutations in them lead to the appearance of normal body parts in unusual places
-Ultrabithorax mutants produce an extra pair of wings

Production of Body Plan
Homeotic gene complexes
-The HOM complex genes of Drosophila are grouped into two clusters
-Antennapedia complex, which governs the anterior end of the fly
-Bithorax complex, which governs the posterior end of the fly
-Interestingly, the order of genes mirrors the order of the body parts they control

Homeotic genes specify body segment identity in Drosophila.
Fig. 14

Homeotic Genes Determine
Specialization of Segments

Antennapedia complex
5 genes that affect the anterior part of the fly
When mutated, legs grow instead of antennae

Homeotic Genes:
Master Regulatory Genes

Homeotic mutation:
Master Regulatory Genes

Halteres into wings
Gilbert, SF (2003) Developmental Biology, 7th ed.
One structure is placed in the position where normally another structure would be (e.g. a pair of wings instead of halteres)

Production of Body Plan
Homeotic gene complexes
-All of these genes contain a conserved 180-base sequence, the homeobox
-Encodes a 60-amino acid DNA-binding domain, the homeodomain
-Homeobox-containing genes
are termed Hox genes
-Vertebrates have 4 Hox gene clusters

Production of Body Plan

Как произошли мухи?
Мухи эволюционировали
из насекомых, имевших 4 крыла,
Насекомые произошли из
членистоногих,
имевших множество ног.
В ходе эволюции у мух
сформировались группы генов:
а) подавляющие развитие ног на
брюшных сегментах
многоножкоподобных предков,
б) подавляющих развитие второй
пары крыльев,
В) формирующие новые
структруры: гальтеры
и брюшные сегменты.

К чему приводит делеция ВХ-С?
Эмбрион развивается до определенной стадии и затем гибнет
Эмбрион имел
проторакальный сегмент, а
все остальные — мезоторакальные
Если бы этот организм выжил до взрослой мухи,
она бы имела 10 пар крыльев и 10 пар ног
Функция гена ВХ-С
- инактивация генов, формирующих ноги и крылья во всех последующих после второго торакального сегментах,
- развитии всех структур на брюшных сегментах

Vertebrates have four Hox complexes,
with about 10 genes each.
They can be aligned in 13 groups of paralogues.
They display colinearity:
Temporal colinearity: genes on one end of the complex are expressed first, those on the other (posterior) end are turned on last.
Spatial colinearity: the more anteriorly expressed genes are in one end, the more posterior ones at the other end of the gene complex.
Anterior Hox genes are activated sequentially by retinoic acid.

Temporal and spatial colinearity: order of Hox genes in DNA follows the antero-posterior body axis.

An identical or very similar nucleotide sequence
Has been discovered in the homeotic genes of both vertebrates and invertebrates

A summary of gene activity during
Drosophila development

Homeobox & Homeodomain
ДНК связывающий модуль
факторов транскрипции
генов дифференцировки
180 пн
60 аминокислотных остатков

Hox Genes and EvoDevo
A= anterior
Group 3
C= Central
P=Posterior

Mutation in HoxD13—synpolydactyly
Extra digits & interphalangeal webbing (hetero)
Similar but more severe & bony malformation
of hands, wrists (Homo)
Loss of function-mild
Gain of function (Polyalanine expansion
in HoxD13) similar to multiple
loss of function-HoxD11,12,13
Polyalanine expansion-
Interfere all three proteins
Semidominant effect

Normal mouse
embryo
Retinoic acid:
loss of many
vertebrae
More retinoic acid:
no posterior region formed
Vitamin A has caused the homeotic HOX genes 1-4 to become expressed in groups of cells that usually do not express these genes. It is well known that high doses of vitamin A taken by women early during pregnancy can disturb the regulation of HOX genes even in the human embryo and cause severe malformations.
Effect of very high doses of retinoic acid on Hox gene expression in
mouse embryos

Hierarchy of genes in Drosophila development
Maternal factor
Development of the number of segments

Hox genes in the Animal Kingdom