MiR-205 is an epithelial-specific microRNA (miR) that supports thymopoiesis. This miR positively regulates Forkhead Box N1 (Foxn1) transcription factor expression. MiR-205 is embedded in a long noncoding RNA (lncRNA), termed MIR205HG in humans. Several lncRNAs that contain miRs have independent functional roles in tissue development and/or regeneration. We characterized the transcriptome assembly of the murine locus containing miR-205 and the larger lncRNA. Conditional knockout mice that harbored a targeted deletion of the proximal region of the lncRNA, with miR-205 sequences retained, were developed. The phenotypes in these mice were compared to those with a miR-205 deficiency. The more severe stress-induced thymic atrophy reported in miR-205-deficient mice, compared to littermate controls, is also evident in lncRNA205 knockout lines. In contrast, a lncRNA205 deficiency results in a smaller mouse stature, which may be coupled with a reduced fat but normal lean mass. These phenotypic differences are explained, in part, by the differential regulation of the lncRNA transcript relative to the pre-mature miR-205. Interferon and IL-22, cytokines released following inflammation, transiently reduce miR-205 while increasing the lncRNA. This is partly determined by the DNp63 transcription factor, which controls miR-205 expression. Gene expression comparisons are being undertaken to reveal how the transcriptome is differentially regulated by these two non-coding RNA species. Further, the mechanism by which these noncoding RNAs regulate Foxn1 is being elucidated. Taken together, these findings suggest both overlapping and independent functions for the lncRNA205 and the embedded miR-205.

Forkhead Box N1 (FoxN1) is an epithelial-specific transcription factor essential for the development of the thymus. Patients with mutations in Foxn1 (OMIM # 600838) are born with a severe T-cell lymphopenia that presents in combination with alopecia and nail dystrophy. The nude mouse, which developed from a spontaneous genetic mutation in Foxn1, phenocopies the human disease. We report on 3 independently identified patients that presented with low to absent circulating T cells. Genetic workup of these patients revealed mutations in Foxn1. Each patient had distinct compound heterozygous mutations that were localized in the distal exons of Foxn1. These were predicted to maintain Foxn1 expression while adversely affect its function. Of significance, each patient had normal hair and skin, without any evidence of nail dystrophy, distinct from previously reported phenotypes. To better define the molecular mechanisms leading to this novel clinical presentation, we used CRISPR/Cas techniques to introduce the corresponding mutations in the mouse Foxn1 sequence. We will present data on the phenotypes of these mice, using intercrosses between individual mutant mice. Comparative transcriptome analyses of fetal thymii from these mice will reveal how the Foxn1 mutations impacts thymic epithelial gene expression and function compared to normal Foxn1. Our findings may lead to better understanding of the role of Foxn1 epithelial cell development and function in both the thymus and skin.

Patients with 22q11.2 deletion syndrome and those with mutations in the Forkhead Box N1(FOXN1) transcription factor (Nude/SCID) can both present with a thymic hypoplasia that results in a severe T cell lymphopenia. In both clinical conditions, the thymic anlage fails to develop properly within the 3rdpharyngeal pouch during embryogenesis. We characterized the development of the thymus in mouse models of 22q11.2 deletion syndrome (22q11.2del) and a new set of mice with mutations in Foxn1that genocopied a SCID patient with novel compound heterozygous mutations in FOXN1. Both sets of mice develop hypoplastic thymic lobes. An analysis of thymopoiesis in embryos revealed distinct development problems. The hypoplastic thymii from the 22q11.2del mice were primarily sized restricted, with normal percentages of all thymocyte subsets apparent. This contrasted a severe deficiency of thymocytes due to the Foxn1mutations, which primarily affected the development and expansion of thymic epithelial cells. Comparative gene expression analyses of e13.5 fetal thymii revealed differentially regulated transcripts that define the basis of the hypoplasia. A dysregulated mesenchymal cell signature was apparent in the 22q11.2del model, which contrasted the epithelial transcript disruption due to the Foxn1mutations. These results suggest different strategies are necessary to correct the thymic tissue abnormalities in patients who present with thymic hypoplasias due to their congenital disorders.

Chromosome 22q11.2 deletion syndrome (22q11.2 ΔS) is the most common microdeletion disorder reported (1/4000). Individuals with this deletion have variable, multi-system disorders including thymic hypoplasia, cardiac anomalies, hypoparathyroidism, and/or dysmorphic facial features. Over 90% of patients have a deletion of 2.4 Mb, which comprises 90 genes, 50% protein coding and the remainder microRNAs, long noncoding RNAs and pseudogenes. The principal cause of the development defects is a haploinsufficiency of the T-box 1 transcription factor (Tbx1). Between 40–60% of patients have some degree of thymic hypoplasia, resulting in systemic T cell lymphopenia. Defects in the thymic stromal tissue is the underlying cause of the hypoplasia. We analyzed the thymic tissue in the mouse models of 22q11.2 ΔS (Df1/+). Comparative transcriptome analyses of hypoplastic and normal-sized lobes derived from the same Df1/+ embryo revealed a signature mRNA expression pattern unique to hypoplastic lobes. Ingenuity pathway analysis uncovered selective pathways compromised in the hypoplastic lobes. Fetal thymic organ culture and reaggregate cultures are currently being used to identify the genes essential for the specification and expansion of the thymic stroma. In addition, normal and hypoplastic thymii, including some from 22q11.2 ΔS patients, are being characterized for the expression of the over- and under-represented genes identified in the mouse model. Findings from these studies may lead to better strategies for improving human thymopoiesis in patients with conditions including 22q11.2 and 10p syndromes, those undergoing chemoablative treatments, and other conditions leading to a thymic hypoplasia and ensuing T cell lymphopenia.