Download Лекция 9. Производные мезодермы, часть 2: эмбриональное

Эмбриональное
кроветворение
Линии мышей, несущих мутации, при которых наблюдается снижение
численности популяции и нарушение миграции кроветворных клеток :
W – мутации в локусе W
Sl (Steel) – мутации в локусе Sl
# локус W несет ген
рецептора тирозинкиназы
(c-Kit)
# локус Sl кодирует его
лиганд (Kit-ligand или Steel
factor – SLF).
Мыши из коллекций Jackson’s Laboratories (США), крупнейшего
центра, хранящего генетические линии лабораторных животных:
Рис.1: у мыши окрашены только радужка глаз и уши.
Рис.2: меланоциты отсутствуют на некоторых участках тела
мышей.
Высокая активность
тирозинкиназы необходима
для нормального процесса
размножения и миграции
популяций, клеток,
мигрирующих в эмбриональный период развития
(ППК, клетки нервного гребня,
кроветворные клетки).
Экспрессия различных типов гемоглобинов в онтогенезе человека
Газообмен
между гемоглобинами
матери и плода
Эмбрион мыши, 15 сут.
кроветворение
Происхождение
кроветворных клеток:
схема на конец 20в.
Эмбрион мыши, 10,5 сут.
Эмбрион мыши, 11,5 сут.
Аorta-gonad-mesonephros region (AGM):
Diagram of the ontogeny of hematopoietic stem cells. Different letters show distinct
hypotheses. Dotted arrows represent alternative migration pathways
Int J Dev Biol. 2009;53(8-10):1529-40.
Diagram of the ontogeny of mesenchymal stem cells. Different arrows colors and letters show distinct hypotheses
Происхождение
эмбриональных HSC
?
Аorta–gonads–mesonephros (AGM):
• subaortic patches (SAPs), located below the aortic floor
• hematopoietic intraaortic clusters (HIACs)
SAP появляются
раньше, чем HIAC
(8.5–9 vs 10 dpc)
10 dpc:
80-100 HSC
(C) Модель появления и миграции предшественников кроветворных клеток:
HSCs (оранжевые) выявляются в области SAP (1), мигрируют по
направлению к аорте (2), затем HSC проникают через дно аорты и участвуют
в формировании кластеров HIAC (3), а затем выходят в кровоток.
Toward a model for emergence
and migration of embryonic
HSCs.
(A) Localization of hemogenicrelated structures, SAPs
(purple), and HIAC (pink)
within a 10.5-dpc embryo.
(B) Relative distribution of
multipotent precursors
(orange), SAPs (purple), and
HIACs (pink) in 8.5- to 12-dpc
embryos.
Whereas GATA-3 SAPs appear
below the aortic floor as soon
as the first multipotent
precursor is generated,
HIACs are present only at the
AGM stage, when the number
of precursors generated
reaches a peak.
Bertrand YJ et al., Proc Natl Acad Sci U S A. 2005 102(1):134-9
Происхождение
эмбриональных HSC
?
Проточная цитометрия и клеточный сортинг
The combinatorial approach undertaken here allowed us to distinguish between
ECs and hematopoietic cells, by GATA-3, CD41, and Flk1 differential expression.
The 3D examination of SAP anatomy also distinguishes SAP cells from ECs
because the CD31 cell clusters located below the aortic floor do not harbor a
lumen and do not connect with the vascular network
Bertrand YJ et al., Proc Natl Acad Sci U S A. 2005 102(1):134-9
Гемангиобласт:
•стенка желточного мешка
•область P-Sp/AGM
Рara-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region
Ангиобласт
HSC
Гемангиобласт?
Дифференцировка гемангиобластов в стенке желточного мешка
Метод клонального анализа для исследования происхождения гемангиобластов
Адгезивные
клетки
Неадгезивные
клетки
Analysis of embryos carrying complementary
DNA of the green fluorescent protein targeted to the
brachyury locus demonstrates that the
haemangioblast is a subpopulation of mesoderm that
co-expresses brachyury (also known as T).
GFP–Bry embryos at E7.5 were dissected into the yolk
sac (YS), anterior (Ant), lateral (L), posterior primitive
streak (PPS) and distal primitive streak (DPS) and
cultured in haemangioblast conditions (numbers of
haemangioblast-derived colonies from fragments)
CD31 (эндотелиальный маркер) –зеленый
Гладкомышечный актин - красный
A model of haemangioblast development in the
mouse embryo. The haemangioblast is a
brachyury+ cell that arises in the primitive streak
and migrates onto the yolk sac where it
differentiates into haematopoietic (H), endothelial
(E) and vascular smooth muscle (VSM)
progenitor cells.
NATURE |VOL 432 | 2 DECEMBER 2004 |www.nature.com/nature
Методы in vitro дифференцировки ЭСК для исследования
ранней спецификации клеточных линий
Notch signaling respecifies the
hemangioblast to a cardiac fate
To efficiently generate cardiomyocytes from embryonic stem (ES) cells
in culture it is essential to identify key regulators of the cardiac lineage
and to develop methods to control them.
Using a tet-inducible mouse ES cell line to enforce expression of a
constitutively activated form of the Notch 4 receptor, we show that
signaling through the Notch pathway can efficiently respecify
hemangioblasts to a cardiac fate, resulting in the generation of
populations consisting of more than 60% cardiomyocytes.
Notch signaling can initiate cardiaclineage specification in part through
the coordinated activation of BMP
signaling and the inhibition of the bcatenin dependent Wnt pathway.
The cardiogenic effects of Notch
signaling were demonstrated in the
normal progression of cardiac
mesoderm (Bry-GFP+/Flk-1–) to
cardiomyocytes and in the dramatic
respecification of hemangioblasts to
cardiovascular progenitors. These
progenitors appear to be similar to
the cardiovascular progenitors
(cardiovascular colony-forming cell;
CV-CFC)
Chen et al VOLUME 26 NUMBER 10 OCTOBER 2008 NATURE BIOTECHNOLOGY
Производные мезодермы:
формирование урогенитальнной системы
Нефротом: смена 3 поколений почек в эмбриогенезе
Формирование метанефроса:
взаимодействие выроста протока мезонефроса и нефрогенной бластемы
Эпителизация
почечных
Канальцев:
Клетки экспрессируют
кадгерины (адгезия
клеток) и ламинин
(формирование
базальной мембраны).
Начало апикально-базальной
поляризации клеток
Формирование
плотных контактов
Dors
Vent
мезонефрос
метанефрос
Эмбрион свиньи, 1 мес.
Положение метанефроса плода человека (поперечный срез)
1
4 5
2
3
самец
самка
6
Первичные половые
клетки (ППК) мыши,
окраска на щелочную
фосфатазу
Четыре гипотезы происхождения ППК
(по Дыбану А.П., 1988)
Красным заштрихованы участки, с которыми
связывают происхождение ППК.
1. Гипотеза половой плазмы:
Цитоплазматические детерминанты, ответственные за
формирование ППК, образуются во время оогенеза.
2. Сегрегационная гипотеза: Половые
детерминанты образуются в цитоплазме зиготы после
оплодотворения. Как и в первом случае, эти
цитоплазматические факторы попадают при
дроблении только в некоторые бластомеры, которые и
являются предшественниками ППК.
3. Гипотеза стволовых клеток: ППК
обособляются во внутренней клеточной массе
бластоцисты или в эпибласте до начала гаструляции
и формирования зародышевых листков.
4. Гипотеза зародышевых листков: ППК
происходят от одного из трех зародышевых листков
(эктодермы, энтодермы или мезодермы)
Выделение линии половых клеток у птиц:
CVG – маркер клеток половаго ряда, гомолог гена Vasa
N. Tsunekawa and others, 2000
Выделение линии половых клеток
у мыши
6,0 dpc
7,0 dpc
Diagram from McLaren (1999).
At 6.0 dpc, a signal (solid arrows) coming from the
extraembryonic ectoderm (blue) predisposes cells in the
proximal layer of the epiblast (brown) towards a germ-line
fate. This whole layer of PGC precursors moves (dashed
arrow) towards the primitive streak and up into the
extraembryonic region.
At 7.0 dpc, the newly formed extraembryonic mesoderm
(gold) is moving across to form the exocoelomic cavity
(white). Some of the PGC precursors stop migrating and
constitute the cluster of cells representing the origin of the
germ cell lineage. This may involve a second (unidentified)
signal or signals (solid arrows). Yellow, visceral endoderm
Diagram reproduced from Saitou et al. (2002), illustrating the
fragilis expression pattern during gastrulation.
(a) Lateral view of an early bud stage embryo with expression of
fragilis. Anterior (A) is to the left and posterior (P) to the right.
Strong expression was observed specifically at the base of the
incipient allantois, the location of nascent PGCs.
(b) fragilis expression from mid bud (farthest left) to early head
fold (farthest right) stages, as viewed from the lateral side. fragilis
expression is strong in the centre (arrowheads)—the site of the
founder PGC cluster. fragilis expression fades at the early head
fold stage (extreme right) when PGCs commence migration.
Arrows indicate developing allantois where fragilis expression is
weak or absent.
(c) Lateral views of pre-streak-stage embryos (6.25 dpc) with
expression of fragilis. Intense signal was observed in proximal
epiblast cells adjacent to the extraembryonic
ectoderm (arrowheads).
(d) fragilis expression from pre-streak (6.0 dpc) (farthest left) to
early bud (farthest right) stages. The initial domain of fragilis
expression in the most proximal epiblast followed by its
movement to the posterior region during gastrulation is shown.
This expression pattern matches with
the observations from clonal analysis for the origin, migration and
segregation of the germ cell lineage.
A. McLaren / Developmental Biology 262 (2003) 1–15
Расположение генов ответственных за дифференцировку на половых хромосомах
Х-хромосома:
Dax1
AR
ATRX
Y-хромосома:
SRY
PAR:
рseudo-autosomal regions;
ATRX: Alpha-thalassemia/mental retardation syndrome X-linked;
AZF: azoospermia factor;
CSF2RA: Colony-stimulating factor 2 receptor alpha;
DAX1: DSS-AHC critical region X chromosome gene 1;
DAZ: Deleted in azoospermia;
FRA-X: Fragile X syndrome;
DMD: Duchenne muscular dystrophy;
GK: Glycerol kinase;
HY: Histocompatibility antigen Y;
IL3RA: Interleukin 3 receptor alpha;
IL9R: Interleukin 9 receptor;
Kal1: Kallmann syndrome 1;
POLA: DNA polymerase alpha;
RBMY: RNA-binding motif protein Y chromosome;
SHOX: Short stature homeo box;
SRY: Sex-determining region Y chromosome;
USP9Y: Ubiquitin-specific protease 9 Y chromosome;
XIST: X inactivation-specific transcript;
ZFX: Zinc finger protein X-linked;
FY: Zinc finger protein Y-linked
http://www.endotext.org/pediatrics/pediatrics7/pediatrics7.html
Структура SRY
Structure of mouse and human SRY protein.
The HMG DNA-binding domain is shown in red and the large glutamine-rich domain of the mouse SRY
COOH terminus is in dark yellow.
Nuclear translocation is mediated by one NLS (pink) at either end of the HMG domain.
The NH2-terminal NLS is recognized and bound by calmodulin (CaM), whereas the COOH-terminal acts via
importin . For both mouse and human SRY, a putative transactivation domain (TAD, light yellow) has been
described. The hinge or bridge region (green) interacts with mouse SRY-interacting protein 1 (SIP1/NHERF2) and the KRAB-only protein, whereas human SRY interacts with SIP-1/NHERF2 via its COOH
terminus.
Sex-reversing mutations in human SRY (marked by asterisks) leading to gonadal dysgenesis or
hermaphroditism are mainly found in the HMG domain.
Гонады: мышь, 12,5 суток развития
• General transcription factors, like LHX1, EMX2
and PAX2, are necessary for intermediate
mesoderm development.
• The gonadal ridge differentiates from the
intermediate mesoderm following the action of SF1,
which is regulated by LHX9 and WT1.
• SRY expression, activated by WT1 and GATA4,
induces testis differentiation, characterized by an
increase in SOX9 and a decrease in DAX1
expression. The effect of SRY overcomes the action
of genes that have been proposed to repress
testicular differentiation, like WNT4, RSPO1, βcatenin and FOXL2.
http://www.endotext.org/pediatrics/pediatrics7/pediatrics7.html
Дифференцировка клеток Сертоли
Differentiation of pre-Sertoli cells
into Sertoli cells. Nonpolarized,
dispersed somatic cells visualized by
SOX9 immunofluorescence (green)
represent pre-Sertoli cells at the24-tail
somite (ts) stage. A few hours later, by
28 ts, these cells become polarized,
forming epithelial aggregates that
assemble into testis cords; at this stage
they are referred to as Sertoli cells.
PECAM-1 counterstaining (red) marks
PGCs and endothelial cells.
Model for cell-autonomous and prostaglandin-mediated upregulation
of Sox9 in pre-Sertoli cells. Sry induces Sox9 cell-autonomously
either via a direct or indirect regulatory mechanism (1). Subsequently,
Sox9 maintains its own expression in an autoregulatory loop
(2). In addition, Sry and/or Sox9 serve to upregulate Pgds (3), which
leads to prostaglandin D2 (PGD2) synthesis (4) and secretion. PGD2 can
act by binding to its receptor DP (5), to upregulate Sox9 expression in
a paracrine, and possibly also an autocrine manner (6). Thus cells that
do not express Sry or fail to reach a threshold of Sry expression can be
induced to upregulate Sox9 and differentiate as Sertoli cells. [Adapted
from Smith et al.
Клетки Сертоли и молекулярные каскады
дифференцировки клеток гонады по мужскому типу
Double-headed arrows, binding to a receptor; colored arrows (blue, red, green), differentiation of
precursor cells into testis-specific cell types; black, bold arrow, gene important for cellular process.
Physiol Rev • VOL 87 • JANUARY 2007
Proposed signaling pathways for AMH. Binding of AMH to its type 2 receptor, AMHR2, probably triggers the
formation of a complex between AMHR2 and one or several candidate type 1 receptors, ALK2, 3 and 6. Activated
type 1 receptor(s) phosphorylate Smad molecules 1, 5 or 8, which then bind to Smad 4 and enter the nucleus to
activate BMP-specific reporter genes XVent2 and Tlx2. Smad6 inhibits this pathway. Putative accessory signaling
pathways include nuclear translocation of ß-catenin and its binding to LEF-1 and induction of NFκB nuclear
binding activity. In the absence of AMH, NF-κB subunits p65/RelA and p50/NF-κB are retained in the cytoplasm by
the inhibitory protein IκBa. AMH releases the subunits from this inhibition and allows them to translocate into the
nucleus. Abbreviations: ALK, activin-like kinase; AMH, anti-Müllerian hormone; AMHR2, AMH receptor type 2;
BMP, bone morphogenetic protein; LEF-1 lymphoid enhancer factor 1; NFκB, nuclear factor κB; Smad, mothers
against decapentaplegic related gene product, TGF-β,
transforming growth factor.
Мезонефрос и закладка гонады
(эмбрион человека, 1,5 мес. , сагиттальный срез)
• Like in the male, general
transcription factors, as LHX1,
EMX2 and PAX2, are
necessary for intermediate
mesoderm development.
• The gonadal ridge differentiates from the intermediate
mesoderm following the action of SF1, which is regulated
by LHX9 and WT1. Gene(s) in the DSS region of the X
chromosome (DAX1, MAGEB, other?), as well as WNT4
and FST should be expressed to antagonize testis
differentiation. RSPO1 and β-Catenin are both anti-testis
and pro-ovary genes, essential for early ovarian
differentiation. Germ cell colonization (dependent on BMP
family members, SCF and its receptor c-kit, WNT4 and
FST) and meiosis (dependent on the existence of two X
chromosomes as well as several factors like DAZLA,
MSH5, STRA8 and DMC1) are essential for fetal ovary
stabilization. A number of other factors are involved in
early folliculogenesis (FOXL2, neurotrophins and
neurotrophin tyrosine kinase receptors)
http://www.endotext.org/pediatrics/pediatrics7/pediatrics7.html
Сайты по теме лекции:
Эмбриональное кроветворение у человека:
http://hematologiya.ru/teoriya/stroenie-kletki.-gemopoehz./gemopoehz-u-ehmbriona-i-ploda.htm
Дифференцировка пола по мужскому и по женскаму типу:
http://www.endotext.org/pediatrics/pediatrics7/pediatrics7.html