Foxn1 evidence
Loss of FOXN1 (as seen in DiGeorge syndrome) absent/hypoplastic thymus severe T cell immunodeficiency
Also mutated in nude mouse strain = abnormal hair growth and failure of thymus development, leading to immunodeficiency
importance of chemokines
Liu et al (2005) = mice deficient in CCL21 or its receptor CCR7 show significantly lower numbers of thymocytes than WT until E14.5
Wurbel et al (2001) = mice deficient in CCR9 (receptor for CCL25) show 3-fold decrease in thymocytes until E17.5
Mouse models implications for humans unknown
Notch/Hh signalling in organogenesis
Seminal experiments by Figueiredo et al (2016) in quail/chicks
• Uncovered role for Notch and Hh crosstalk in thymus and parathyroid development = regulate temporal and spatial dynamics of morphogenesis
Thymus and parathyroid develop from 3PP and 4PP in avians
Dissected 3rd and 4th pharyngeal arches from quail embryos at E3 and grew in culture medium (in vitro) for 48hr investigated roles of Notch and Hh signalling by ectopic administration of pharmacological inhibitors = examined gene expression by qRT-PCR
Then grafted explants onto chorioallantoic membrane of chicken embryo (provides nutrients) for in ovo development fully formed organs obtained after 10 days
Major findings
- Impairing Notch signalling with 2 different inhibitors significantly reduced expression of FOXN1 and PAX1 (thymus), and Gmc2 and Pth (parathyroid), in 3/4PP = suggests that Notch signalling required for thymic epithelium specification and early stages of parathyroid epithelium differentiation
- When transferred onto CAM, explants could still form thymic organs = suggests that absence of Notch at early stage delays thymic specification but doesn’t block it entirely
- Abolishing Hh signalling (modulated by Notch) significantly reduced Gata3/Gcm2 and Lfng expression domains at M/A and M/P territories, and expanded Foxn1 expression domain from dorsal tip into more P/M region of pouch, expressing Lfng
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• Replicated findings in vivo = either injecting Notch inhibitor, or inserting beads soaked in Hh inhibitors, into quail embryos
• Limitations of this method
- Suboptimal accessibility of pharmacological reagents to explants = inhibited investigation of effects of Notch inhibition at later stages of development = limited analysis of explant late-stage development
TGF-B KO
• TGF-B = promotes differentiation of cortical epithelium, over medullary
- KO mice show significant overexpansion of medullary TECs and exaggerated negative selection evidence that TGF-B is required to restrain medullary progenitor differentiation
crosstalk between thymocytes and stroma
• Van Evikj et al (2000)
- Compared thymus architecture of Tge26 mice (overexpression of human CD3e gene = block at stage of CD44+ CD25- cells) to Rag KO mice (block at later CD44- CD25+ stage) with SEM and TEM
- Tge26 mice – small thymus, low cellularity, heavily disturbed C+M
- Rag KO mice = increased cellularity, well-established cortex, absent medulla
- Transplanting Tge26 with RAG KO BM converts thymic phenotype to that of RAG null mice transplanting these chimeras with WT bone marrow restores thymic cellularity completely
- Supports argument for crosstalk regulating creation of thymic microenvironments in stepwise fashion = thymic epithelium is very plastic
first evidence of single lineage hypothesis
• First suggested by Le Douarin et al (1975)
- Generated chick-quail chimeras by transplanting pharyngeal endoderm from quail embryos before colonisation by lymphocytes into body cavity of 3do chick
- Grafted tissue developed into thymus = T cells were of chick origin, cTEC and mTEC cells were of quail origin (characterised by single centronuclear condensation vs network of lightly stained chromatin seen in chick TEC)
- Evidence that purified pharyngeal endoderm is sufficient to generate epithelial component of both compartments
endodermal origin for all TECs
• Gordon et al (2004) = implied endodermal origin for all TEC
- Used whole-embryo culture, labelled pharyngeal ectoderm of E10.5 mouse embryos with cell-tracker fluorescent dye CMFDA
- Found that thymic primordium separated from labelled ectoderm = no labelled cells present in thymus of any embryos analysed
- Transplantation of isolated 3rd pharyngeal pouch endoderm under kidney capsule of a nude mouse resulted in formation of an entire functional thymus = normal histological appearance, staining for pan-cytokeratin (epithelial cells), MHC class II (TECs), 4F1 and anti-cytokeratin 14 in regions corresponding to cortex and medulla, flow cytometry revealed presence of DN, DP and SP thymocytes
MTS24 stem cell studies
• Gill et al (2002)
- Showed that foetal thymus contains population of early stem cells that can give rise to both mTEC and cTEC
- Defined by expression of MTS24
- Isolated thymus, dispersed into single cell suspension and purified MTS24+ cells using FACS transplanted under kidney capsule of nude mice gave rise to entire functional thymus, with normal medulla and cortex, progenitors and ability to sustain positive and negative selection
- MTS24- cells could not sustain development of thymus
clonogenic assays = evidence of SLH
Rossi et al (2006) = provided definitive proof at clonogenic level that stem cells can create both cTEC and mTEC, and that only one lineage is required for thymus
• Used mice that constitutively expressed YFP under ubiquitous promoter (all cells expressed YFP)
• Took embryos at E12 thymus rudiment only just become colonised by haematopoietic elements = thymus is mainly epithelial cells
• Dissected developing thymus rudiments and dispersed in single cell suspension = found population of early stem cells expressing YFP and EpCAM1 (co-expressed by MTS24)
• Clonogenic assays = took single cell and injected into foetal thymus dissected from E14 embryo (when thymus has separated) cultured in vitro for 1 week (thymus can continue to develop normally in culture and can support T cell repertoire selection = good model for in vivo development)
• Sectioned and stained thymus for YFP
- Found multiple progeny of single injected cell in equal proportions in cortex and medulla
- Co-labelling experiments showed that cells expressing YFP also expressed markers of cTEC (Ly51) or mTEC (MTS10)
mesenchyme contribution
Jenkinson et al (2006)
• Generated reaggregate thymi = isolated mTEC and cTEC, mixed together and produced pellet of cells in centrifuge that coalesced to form discrete structure that could be transplanted under the kidney capsule of the nude mouse
• Some included added mesenchyme, some did not
• Those including mesenchyme formed entire thymi = medulla and cortex, attracted early progenitors from bone marrow and supported repertoire selection (contained both double negative and positive cells, and CD4 and CD8 positive cells)
• Those without mesenchyme produced much smaller thymus, still able to support normal selection but yield of thymocytes was much smaller (around 10% of those with mesenchyme) = indicates role of mesenchyme for stimulating progenitor proliferation
cervical thymus
• Terszowski et al (2006)
- One or more thymi develop in the cervical region of mice and grow to the size of cervical lymph nodes (always assumed to be lymph nodes not thymi!)
- Showed that cells of cervical thymi expressed GFP under Foxn1 promoter, as seen in thoracic thymus
- Cervical thymi are similar in structure to thoracic thymus, and are fully functional = can be divided into cortex and medulla, epithelial cells stain with same markers, sustain normal positive and negative selection
unanswered questions about cervical tolerance
Unanswered questions
• Why does the cervical thymus develop neonatally and not in embryonic period?
• Do they still develop from the 3rd pharyngeal pouch, or something else?
• Which embryonic layers contribute?
• Is there any qualitative difference between T cells that differentiate in cervical and thoracic thymus?
• Does cervical thymus undergo involution?
• How do we interpret a body of experiments on T cell tolerance that depend on the use of thymectomy?
importance of CXCL13 in lymph node organogenesis
Van de Pavert et al (2009) = importance of CXCL13
• Mutant embryos lacking CXCL13 or receptor CXCR5 do not exhibit initial clustering of LTi cells and lack all lymph node anlagen at E14.5
• BUT adult mice have mesenteric + cervical lymph nodes = suggests that development of these nodes can be partly restored later in absence of CXCL13 signals
• Evidence that CXCL13 precedes LTBR signalling no differences in CXCL13 in WT and LTA KO mice at E12.5-E13.5
evidence that RA might be neuron-derived
• Sockanathan et al (1998)
- Brachial and lumbar motor neurons express RALDH2 from E12.5 when lymph node formation is initiated (RT-PCR and IHC)
- Neurons are paracrine source of RA for maintenance of lateral motor column neurons might also provide RA to brachial, axillary and inguinal lymph nodes
- Correlative evidence = just because they secrete RA doesn’t mean that RA contributes to lymph node development
evidence for cross-antagonism in Lti production
• Boos et al (2007)
- ID2 KO mice lack LTi cells and secondary lymphoid tissues
- Combining ID2 KO with E2A KO restores LTi production and development of lymph nodes and Peyer’s patches = deletion of E2A overcomes need for ID2, generation of LTi cells dependent on balance of two molecules
evidence that LTi not required for tertiary lymphoid tissue development
Cupedo (2011)
- Lymphoid neogenesis does not require LTi cells, the requirement for which is replaced by IL-17 secreted by T cells in response to TLR ligands such as LPS
- IL-17 drives secretion of CXCL13 and CCL19 by stromal cells which recruit and organise lymphocytes
