Development of pharyngeal pouches

endoderm-derived glands

Thymus: from organogenesis to involution

Rita Zilhão

ENCONTRO

FCUL, 4 February , 2013 Unidade de Organogénese (IHBD-FMUL/IMM)

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

Faculdade de Medicina de Lisboa

Instituto de Histologia e Biologia do Desenvolvimento

Instituto de Medicina Molecular

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).

Pharyngeal pouches (PP) endoderm-derived glands

Thymus 3rd PP, E11.5 (mouse);

3rd and 4th PP, E4 (chicken)

(PA)

Early phase

Mesenchymal-epithelial

interactions

Late phase

Foxn1

Epithelial-LPC

interactions

TEC

LPC Ect

od

erm

En

do

derm

PA1

PA2

PA3

PA4 3rd PP

4th PP

Adult thymus

Thymus organogenesis

Thymocyte-independent Thymocyte-dependent

E4.5

Isolated 3/4PP endoderm

cTEC

mTEC

(AIRE)

Function: Production of mature T-Lymphocytes

Notch1, 2

Delta1, 3, 4

Serrate1, 2

Hairy1 Hes5.1 Hes6.1 Intracellular

domain of Notch (ICN)

MAML

]

Modulation of Notch signals:

ICN: Gain-of-function

DNMAML: Loss-of-function (Maillard et al. 2004)

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).

Jagged 1, 2

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 epithelial-

mesenchymal 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 growth-

factors soaked-beads

“Tol2-mediated gene transfer” + “tetracycline-dependent conditional expression”

Thymus organogenesis

Sato et al. 2007

Vector system

Thymus development In vitro /in vivo assays

1. Isolation of 3/4PP endoderm

(Quail E3)

48h

3. Organotypic culture

cE8

4. Graft into CAM

5. Explant development in CAM

C.

QCPN

2. Endoderm electroporation

D.

+ doxy

10 days

3. Isolation somatopleural mesenchyme

(Chicken E2.5)

System of vectors: genetic modification of isolated tissues

ICN ou DNMAML

Thymus organogenesis Modulation of Notch signaling in thymic epithelium development

E5-5.5 E4 E3 E2,5

Thymocyte-independent Thymocyte-dependent

E7

Foxn1 expression Aire expression

Development of TECs in quail

Analysis of Aire expression and thymic

compartments formation Dox

Dox

Dox

Analysis of Foxn1 expression and thymus development

Analysis of thymic epithelium colonization by LPCs

Modulation of Notch signaling in vivo assay

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

Thymus involution

With age there is a marked increase in susceptibility to infections

and diseases (ex. autoimune disesases).

Human model

The three events associated with age-related thymic involution

1. A reduction in numbers and intrinsic defects in hematopoietic stem cells (HSCs)

2. Loss of thymic epithelial cells (TECs) and deterioration of stromal microenvironment

3. Extrinsic circulating factors affecting the aged microenvironment, e.g. alterations in

hormones/growth factors/cytokines

Thymus involution

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

Human model

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)