Bleul and Boehm, 2005 Bleul C.C.

Boehm T. BMP signaling is required for normal thymus development.

Meier et al., 1999 Meier N.

Dear T.N.

Boehm T. Whn and mHa3 are components of the genetic hierarchy controlling hair follicle differentiation.

Nakagawa et al., 2013 Nakagawa S.

Gisselbrecht S.S.

Rogers J.M.

Hartl D.L.

Bulyk M.L. DNA-binding specificity changes in the evolution of forkhead transcription factors.

Schlake et al., 1997 Schlake T.

Schorpp M.

Nehls M.

Boehm T. The nude gene encodes a sequence-specific DNA binding protein with homologs in organisms that lack an anticipatory immune system.

Figure 3 Foxn4-Driven Thymopoiesis in Mice Show full caption (A) Schematic of transgenic expression of Foxn1 or Foxn4 in TECs of Foxn1-deficient mice. In the absence of FOXN1 function, TECs do not differentiate (dotted line); transgenic expression of Foxn1 (red oval) or Foxn4 (blue oval) in the Foxn1-deficient background restores thymopoiesis. (B) Expression of transgenes as revealed by RNA in situ hybridization with Foxn1- and Foxn4-specific probes. Note the small thymic rudiment in nontransgenic Foxn1−/− mice that becomes considerably larger in the transgenic animals; Foxn4 expression is detectable only in the Foxn4 transgenic mice. Scale bar, 100 μm. (C) Expression of Foxn1 and Foxn4 relative to β-actin in purified TECs of adult mice as detected by quantitative RT-PCR. The primers used for quantitative PCR also detect the mutant Foxn1 transcript. (D) Histological sections of thymic rudiments/thymi of adult mice (hematoxylin and eosin staining). Scale bar 100 μm. (E) Flow cytometric profiles of thymocytes of adult mice stained with antibodies directed against the coreceptors CD4 and CD8. See also Figure S3 and Tables S1–S3

Figure 4 Thymopoiesis in Adult Transgenic Mice Show full caption (A) Thymocyte populations are shown. DP, thymocytes expressing both CD4 and CD8; DN, thymocytes expressing neither CD4 nor CD8; CD4SP, thymocytes expressing only CD4; CD8SP, thymocytes expressing only CD8. (B) Splenocyte populations in adult transgenic mice of the indicated genotypes. (C) Characterization of lymph node cells in adult transgenic mice of the indicated genotypes. Total cellularity of lymph nodes (top panel) and proportions of lymph node CD4+ (middle panel) and CD8+ (bottom panel) T cells. (D) TCR Vß usage in adult transgenic mice of the indicated genotypes as determined by flow cytometry. Each bar represents the percentage of CD4+ (left) and CD8+ (right) splenic T cells from an individual mouse that utilize the indicated Vß segment. (E) In vitro stimulation of lymph node cells of the indicated phenotypes with anti-CD3. Viable cells were identified as hydroxystilbamidine negative, and the proportion of divided cells (CD4+ or CD8+) was determined by CSFE content. (F) Anti-NP immunoglobulin G1 titers after immunization of adult mice with CGG-NP in arbitrary units (a.u.). (G) Fraction of FOXP3+ Treg cells among lymph node CD4+ T cells. The numbers of mice analyzed per genotype are indicated. See also Figure S4 and Table S3

To model the hypothetical primordial situation of Foxn4-driven thymopoiesis in mice, we replaced Foxn1 by Foxn4 in the mouse thymic epithelium ( Figure 3 A). To this end, Foxn1-deficient mice were supplied with transgenes in which either Foxn1 (as a control) or Foxn4 cDNAs were expressed in TECs, under the regulatory elements of the Foxn1 gene () ( Figure S3 B). Under these conditions, Foxn4 becomes expressed both in the thymic epithelium ( Figures 3 B and 3C) and in the hair follicle ( Figure S3 C), an additional site of Foxn1 expression in the mouse (). Restoration of normal hair growth not only in Foxn1;Foxn1:Foxn1 controls but also in Foxn1Foxn1:Foxn4 transgenic mice ( Figure S3 C) indicates that Foxn1 promoter elements are capable of regulating transgene expression in a physiologically normal manner and that the expression levels of Foxn4 are sufficient to achieve coordinate regulation of keratinocyte differentiation in the skin. This observation further supports the notion that the FOXN4 transcription factor activates most, if not all, FOXN1-dependent target genes, which is compatible with their similar DNA binding specificities (). With respect to the thymus, we found a normal histological structure in control Foxn1;Foxn1:Foxn1 transgenic mice ( Figure 3 D) and a normal pattern of T cell development ( Figure 3 E). Surprisingly, Foxn4 expression also restored failing thymopoiesis in Foxn1mice. In Foxn1;Foxn1:Foxn4 transgenic mice, the thymus is characterized by regions of varying cellular density, reminiscent of cortical and medullary areas in the wild-type thymus, although some cystic regions persist ( Figure 3 D). Despite these histological perturbations, the major thymocyte populations were present ( Figure 3 E). In Foxn1;Foxn1:Foxn4 transgenic mice, the absolute number of thymocytes attained up to 30% of normal levels, albeit with considerable variation among individual mice ( Figure 4 A); one notable difference to wild-type or Foxn1;Foxn1:Foxn1 transgenic mice is the considerable increase in the proportion of CD4/CD8cells ( Figure S4 A), which will be addressed in more detail below. The thymopoietic activity in reconstituted transgenic mice led to the presence of large numbers of peripheral T cells in the spleen ( Figure 4 B) and lymph nodes ( Figure 4 C), reaching almost normal levels in Foxn1;Foxn1:Foxn1 transgenic mice and subnormal levels in Foxn1;Foxn1:Foxn4 mice ( Figures 4 B and 4C), with some indication of oligoclonality of the T cell repertoire in the latter group ( Figure 4 D; Figure S4 B). As judged by several criteria, the peripheral T cell compartments in transgenic mice appear to be functionally competent. For instance, T cells isolated from the lymph nodes of reconstituted mice proliferated in response to TCR crosslinking in vitro ( Figure 4 E). In accordance with the restoration of thymopoiesis, the majority of Foxn1;Foxn1:Foxn4 mice mounted hapten antibody responses ( Figure 4 F), in line with the varying extent of thymopoietic activity ( Figures 4 A–4D). Foxn1;Foxn1:Foxn4 mice had a normal lifespan and did not exhibit overt autoimmune phenomena, compatible with the presence of FOXP3+ regulatory T cells ( Figure 4 G). Collectively, these data indicate that T cell immunity was restored in Foxn1mice expressing Foxn4 in the thymic epithelium. Together, these results support the notion that, when expressed at sufficiently high levels in thymic epithelial cells (TECs), FOXN4 can at least partially replace FOXN1 in supporting thymopoietic functions not only in fish but also in mice.