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During development, cortical c and medullary m thymic epithelial cells TEC arise from the third pharyngeal pouch endoderm. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction.

Using potency analyses we also establish that most E Finally we show that overnight culture causes most if not all E Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells. T cell repertoire development, called thymopoiesis, depends on dynamic interactions between the developing T cells thymocytes and the stromal compartment of the thymus 1 , 2.

This compartment is complex, comprising epithelial, mesenchymal, vascular and haematopoietic components organized into two regions, the cortex and the medulla, which support different aspects of thymopoiesis 3 — 5.

During development TEC arise from the endoderm of the third pharyngeal pouches 3PP , which also generate the parathyroid glands. When differentiation of the endodermal cells in the thymus domain of the 3PP is blocked, for instance by the lack of a functional allele encoding the transcription factor Forkhead Box N1 FOXN1 , a functional thymus able to support T cell lineage commitment and subsequent thymopoiesis cannot be generated 15 , 16 ; FOXN1 is required throughout life for normal differentiation of both cTEC and mTEC 17 and is regarded as a master regulator of TEC differentiation 15 — In addition, an elegant clonal analyses using single cell reversion of a null allele of Foxn1 has shown that bipotent TEPC can exist in vivo 18 , and a single cell transplantation analysis also suggested most cells in the early fetal thymus are bipotent TEPC Based on these observations, a serial progression model of TEC differentiation has been proposed Testing this model has been hampered by difficulties related to identification of cTEC-restricted sub-lineage specific progenitor TEC in the fetal thymus, due to shared expression of surface antigens between this presumptive cell type and the presumptive common TEPC 20 , 22 , 23 , although cTEC-restricted progenitors clearly exist in the postnatal thymus In contrast, the presence of functionally validated mTEC-restricted progenitors has been detected from day However, lineage divergence can also occur downstream of FOXN1 expression Therefore, consistent with phenotypic analyses 17 , 26 , 28 , but in contradiction to the serial progression model, they predict the presence of mTEC-fated TEC within the thymic primordium prior to E Early thymus organogenesis has recently begun to be mapped using scRNA sequencing 35 , 36 and in vivo barcoding In particular, Magaletta and colleagues found that TEC could be clustered into two populations at E Furthermore, in vivo barcoding initiated by Foxn1Cre supported the presence of cTEC-fated TEPC in the fetal thymus but did not reveal a common or mTEC-restricted progenitor active in the fetal thymus; indeed, this study did not provide information regarding the source of mTEC during fetal development, although both mTEC-biased and common TEC progenitor activities were observed postnatally Collectively therefore, these data also support a model in which cTEC and mTEC fates initially diverge early in thymus organogenesis.