The forkhead transcription factor Foxn1 is indispensable for thymus development, but the mechanisms by which it mediates thymic epithelial cell (TEC) development are poorly understood. To examine the cellular and molecular basis of Foxn1 function, we generated a novel and revertible hypomorphic allele of Foxn1. By varying levels of its expression, we identified a number of features of the Foxn1 system. Here we show that Foxn1 is a powerful regulator of TEC differentiation that is required at multiple intermediate stages of TE lineage development in the fetal and adult thymus. We find no evidence for a role for Foxn1 in TEC fate-choice. Rather, we show it is required for stable entry into both the cortical and medullary TEC differentiation programmes and subsequently is needed at increasing dosage for progression through successive differentiation states in both cortical and medullary TEC. We further demonstrate regulation by Foxn1 of a suite of genes with diverse roles in thymus development and/or function, suggesting it acts as a master regulator of the core thymic epithelial programme rather than regulating a particular aspect of TEC biology. Overall, our data establish a genetics-based model of cellular hierarchies in the TE lineage and provide mechanistic insight relating titration of a single transcription factor to control of lineage progression. Our novel revertible hypomorph system may be similarly applied to analyzing other regulators of development.

Thymic involution is central to the decline in immune system function that occurs with age. By regenerating the thymus, it may therefore be possible to improve the ability of the aged immune system to respond to novel antigens. Recently, diminished expression of the thymic epithelial cell (TEC)-specific transcription factor Forkhead box N1 (FOXN1) has been implicated as a component of the mechanism regulating age-related involution. The effects of upregulating FOXN1 function in the aged thymus are, however, unknown. Here, we show that forced, TEC-specific upregulation of FOXN1 in the fully involuted thymus of aged mice results in robust thymus regeneration characterized by increased thymopoiesis and increased naive T cell output. We demonstrate that the regenerated organ closely resembles the juvenile thymus in terms of architecture and gene expression profile, and further show that this FOXN1-mediated regeneration stems from an enlarged TEC compartment, rebuilt from progenitor TECs. Collectively, our data establish that upregulation of a single transcription factor can substantially reverse age-related thymic involution, identifying FOXN1 as a specific target for improving thymus function and, thus, immune competence in patients. More widely, they demonstrate that organ regeneration in an aged mammal can be directed by manipulation of a single transcription factor, providing a provocative paradigm that may be of broad impact for regenerative biology.

SummaryA central goal of regenerative medicine is to generate transplantable organs from cells derived or expanded in vitro. Although numerous studies have demonstrated production of defined cell-types in vitro1, creation of a fully intact organ has not been reported. The transcription factor Forkhead box N1 (FOXN1) is critically required for development of thymic epithelial cells (TECs)2,3 a key cell-type of the thymic stroma4. Here, we show that enforced Foxn1 expression is sufficient to reprogramme fibroblasts into functional TECs, an unrelated cell-type across a germ-layer boundary. These Foxn1-induced TECs (iTECs) supported efficient development of both CD4+ and CD8+ T cells in vitro. Upon transplantation, iTEC established a complete, fully organized and functional thymus, that contained all of the TEC sub-types required to support T cell differentiation and populated the recipient immune system with T cells. iTEC thus demonstrate that cellular reprogramming approaches can be used to generate an entire organ, and open the possibility of widespread use of thymus transplantation to boost immune function in patients.

FoxG1 is an evolutionarily conserved, winged-helix transcriptional repressor that maintains progenitor cells in the vertebrate forebrain. How the activity of FoxG1 is regulated is not known. Here, we report that in the developing Xenopus and mouse forebrain, FoxG1 is nuclear in progenitor cells but cytoplasmic in differentiating cells. The subcellular localisation of FoxG1 is regulated at the post-translational level by casein kinase I (CKI) and fibroblast growth factor (FGF) signalling. CKI phosphorylation of Ser 19 of FoxG1 promotes nuclear import, whereas FGF-induced phosphorylation of Thr 226 promotes nuclear export. Interestingly, FGF-induced phosphorylation of FoxG1 is mediated Akt kinase (also known as protein B kinase, PKB) kinase, rather than the MAPK pathway. Phosphorylation of endogenous FoxG1 is blocked by CKI and Akt inhibitors. In the mouse olfactory placode cell line OP27, and in cortical progenitors, increased FGF signalling causes FoxG1 to exit the nucleus and promotes neuronal differentiation, whereas FGF and Akt inhibitors block this effect. Thus, CKI and FGF signalling converge on an antagonistic regulation of FoxG1, which in turn controls neurogenesis in the forebrain.

Recently naive human pluripotent stem cells (hPSCs) have been described that relate to an earlier stage of development than conventional hPSCs. Naive hPSCs remain challenging to generate and authenticate, however. Here we report that Sushi Containing Domain 2 (SUSD2) is a robust cell-surface marker of naive hPSCs in the embryo and in vitro. SUSD2 transcripts are enriched in the pre-implantation epiblast of human blastocysts and immunostaining shows localization of SUSD2 to KLF17-positive epiblast cells. SUSD2 mRNA is strongly expressed in naive hPSCs but is negligible in other hPSCs. SUSD2 immunostaining of live or fixed cells provides unambiguous discrimination of naive versus conventional hPSCs. SUSD2 staining or flow cytometry enable monitoring of naive hPSCs in maintenance culture, and their isolation and quantification during resetting of conventional hPSCs or somatic cell reprogramming. Thus SUSD2 is a powerful non-invasive tool for reliable identification and purification of the naive hPSC phenotype.

SummaryIn contrast to conventional human pluripotent stem cells (hPSCs) that are related to post-implantation embryo stages, naive hPSCs exhibit features of pre-implantation epiblast. Naive hPSCs are established by resetting conventional hPSCs, or are derived from dissociated embryo inner cell masses. Here we investigate conditions for transgene-free reprogramming of human somatic cells to naive pluripotency. We find that Wnt inhibition promotes RNA-mediated induction of naive pluripotency. We demonstrate application to independent human fibroblast cultures and endothelial progenitor cells. We show that induced naive hPSCs can be clonally expanded with a diploid karyotype and undergo somatic lineage differentiation following formative transition. Induced naive hPSC lines exhibit distinctive surface marker, transcriptome, and methylome properties of naive epiblast identity. This system for efficient, facile, and reliable induction of transgene-free naive hPSCs offers a robust platform, both for delineation of human reprogramming trajectories and for evaluating the attributes of isogenic naive versus conventional hPSCs.

Multiple GnRH receptors are known to exist in nonmammalian species, but it is uncertain which receptor type regulates reproduction via the hypothalamic-pituitary-gonadal axis. The teleost fish, Astatotilapia burtoni, is useful for identifying the GnRH receptor responsible for reproduction, because only territorial males reproduce. We have cloned a second GnRH receptor in A. burtoni, GnRH-R1(SHS) (SHS is a peptide motif in extracellular loop 3), which is up-regulated in pituitaries of territorial males. We have shown that GnRH-R1(SHS) is expressed in many tissues and specifically colocalizes with LH in the pituitary. In A. burtoni brain, mRNA levels of both GnRH-R1(SHS) and a previously identified receptor, GnRH-R2(PEY), are highly correlated with mRNA levels of all three GnRH ligands. Despite its likely role in reproduction, we found that GnRH-R1(SHS) has the highest affinity for GnRH2 in vitro and low responsivity to GnRH1. Our phylogenetic analysis shows that GnRH-R1(SHS) is less closely related to mammalian reproductive GnRH receptors than GnRH-R2(PEY). We correlated vertebrate GnRH receptor amino acid sequences with receptor function and tissue distribution in many species and found that GnRH receptor sequences predict ligand responsiveness but not colocalization with pituitary gonadotropes. Based on sequence analysis, tissue localization, and physiological response we propose that the GnRH-R1(SHS) receptor controls reproduction in teleosts, including A. burtoni. We propose a GnRH receptor classification based on gene sequence that correlates with ligand selectivity but not with reproductive control. Our results suggest that different duplicated GnRH receptor genes have been selected to regulate reproduction in different vertebrate lineages.

Thymus function requires extensive cross-talk between developing T-cells and the thymic epithelium, which consists of cortical and medullary TEC. The transcription factor FOXN1 is the master regulator of TEC differentiation and function, and declining Foxn1 expression with age results in stereotypical thymic involution. Understanding of the dynamics of Foxn1 expression is, however, limited by a lack of single cell resolution data. We have generated a novel reporter of Foxn1 expression, Foxn1G, to monitor changes in Foxn1 expression during embryogenesis and involution. Our data reveal that early differentiation and maturation of cortical and medullary TEC coincides with precise sub-lineage-specific regulation of Foxn1 expression levels. We further show that initiation of thymic involution is associated with reduced cTEC functionality, and proportional expansion of FOXN1-negative TEC in both cortical and medullary sub-lineages. Cortex-specific down-regulation of Foxn1 between 1 and 3 months of age may therefore be a key driver of the early stages of age-related thymic involution.