Development of pharyngeal pouches
endoderm-derived glands
Thymus: from organogenesis to involution
Unidade de Organogénese (IHBD-FMUL/IMM)
Rita Zilhão
ENCONTRO
FCUL, 4 February , 2013
Faculdade de Medicina de Lisboa
Instituto de Histologia e Biologia do Desenvolvimento
Instituto de Medicina Molecular
U. Organogénese
Hélia Neves
Rita Zilhão
Marta Figueiredo
Carlota Lucena
Ana sofia Santos
Joana Clara Silva
André Travessa
Adriano Carvalho
Vitor Proa
U. Biologia Hematopoiética
António Cidadão
Isabel Alcobia
Pedro Moreira
Pharyngeal pouches (PP) endoderm-derived glands
Thymus: differentiation of T lymphocytes;
Parathyroid glands: production of parathyroid hormone (PTH) and control of the
calcium amount in blood and bones;
C-cells: production of calcitonin (a subtype of thyroid cells).
(PA)
Thymus 3rd PP, E11.5 (mouse);
3rd and 4th PP, E4 (chicken)
Thymus organogenesis
Late phase
Early phase
PA1
PA2
PA3
PA4
Thymocyte-dependent
Ectoderm
Endoderm
Thymocyte-independent
Adult thymus
cTEC
LPC
Foxn1
3rd PP
4th PP
Mesenchymal-epithelial
interactions
TEC
Epithelial-LPC
interactions
mTEC
(AIRE)
Function:
Production of mature T-Lymphocytes
E4.5
Isolated 3/4PP endoderm
The role Notch signaling in thymus organogenesis
Notch signaling pathway is important for cell-cell communication, involving gene
regulation mechanisms that control several aspects of organogenesis (cell fates, cell
numbers and cell position).
MAML
]
Notch1, 2
Intracellular
domain of Notch
(ICN)
Delta1, 3, 4
Jagged 21, 2
Serrate1,
Hairy1
Hes5.1
Hes6.1
Modulation of Notch signals:
ICN: Gain-of-function
DNMAML: Loss-of-function
(Maillard et al. 2004)
Thymus organogenesis
The avian model
•
Easy access to and manipulation of chicken embryos from incubated eggs
•
Isolation of embryonic tissues (microdissection)
•
Tissue cultures:
– Heterospecific (quail-chick) association of tissues to study epithelialmesenchymal interactions during development
– Organotypic system: explanted tissues grown and organ formation in
corioallantoic membrane (CAM) of chick embryos
•
Genetic modification of embryonic tissues using a system of vectors
•
In vivo modulation of genetic pathways during avian development using growthfactors soaked-beads
Thymus organogenesis
Vector system
“Tol2-mediated gene transfer” + “tetracycline-dependent conditional expression”
Sato et al. 2007
Thymus development
In vitro /in vivo assays
1. Isolation of 3/4PP endoderm
System of vectors: genetic modification of isolated tissues
ICN ou DNMAML
(Quail E3)
2. Endoderm electroporation
3. Isolation somatopleural mesenchyme
3. Organotypic culture
(Chicken E2.5)
48h
4. Graft into CAM
C.
D.
10 days
+ doxy
cE8
5. Explant development in CAM
QCPN
Thymus organogenesis
Modulation of Notch signaling in thymic epithelium development
Development of TECs in quail
E2,5 E3
E5-5.5
E4
E7
Thymocyte-independent
Thymocyte-dependent
Foxn1 expression
Aire expression
Modulation of Notch signaling in vivo assay
Analysis of Foxn1 expression and thymus development
Analysis of thymic epithelium colonization by LPCs
Dox
Analysis of Aire expression and thymic
compartments formation
Dox
Dox
Thymus involution
Human model
Normal conditions
• Rapidly declines in function to <5% of its maximal capacity after puberty
• In healthy individuals this level of residual thymic function is often sufficient
With age there is a marked increase in susceptibility to infections
and diseases (ex. autoimune disesases).
Thymus involution
Human model
The three events associated with age-related thymic involution
1.
2.
3.
A reduction in numbers and intrinsic defects in hematopoietic stem cells (HSCs)
Loss of thymic epithelial cells (TECs) and deterioration of stromal microenvironment
Extrinsic circulating factors affecting the aged microenvironment, e.g. alterations in
hormones/growth factors/cytokines
Future studies
Modifications of thymic epithelial cells along the first years of life (Descriptive approach)
•Gene expression study of thymic epithelial cells
•Correlation expressions patterns in TECs/thymic age
Role of notch signaling in thymic epithelium homeostasis (Functional approach)
•Modulation of Notch signaling in thymic epithelial cells
“Recreating” thymic environment
Projects
•
•
•
2012/13
–
The role of Notch signaling in thymic and parathyroid glands organogenesis (Joana Silva, mBMG)
–
Bioinformatics approach on the evolution of genes related with the origin of the thymus (Bruno Costa,
mBBC)
–
Hox-code in thymus identity (Ana Sofia Santos, mBED)
–
Role of Notch signalling on the differentiation of early lymphoid progenitors cells: a view throughout the
development of the embryonic chicken thymus (Pedro Moreira, mGMB, FCT/UNL)
–
Avaliação morfológica e molecular da junção córtico-medular do timo humano, ao longo dos primeiros anos
de vida: implicações na involução tímica (André Travessa, GAPIC)
–
The role of Notch-signaling in avian thymus and parathyroid glands development (Marta Figueiredo,
Doutoramento/FMUL)
2011/12
–
Embryonic thymic epithelium differentiation in chicken: Study of molecular signals involved in lymhoid
progenitor cells colonization (Carlota Lucena, mBMG)
–
Role of Notch signaling in the differentiation of endocrine C-cells during embryogenesis (Rita Zilhão, ½
sabática, DBV/FCUL) (Programa PESSOA 2010-11, Proc. 441.00)
2010/11
–
The role of Notch signaling in thymic epithelium development (Marta Figueiredo, mBED)