Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion.

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

TSHR Variant Screening and Phenotype Analysis in 367 Chinese Patients With Congenital Hypothyroidism.

Background: Genetic defects in the human thyroid-stimulating hormone (TSH) receptor (TSHR) gene can cause congenital hypothyroidism (CH). However, the biological functions and comprehensive genotype-phenotype relationships for most TSHR variants associated with CH remain unexplored. We aimed to identify TSHR variants in Chinese patients with CH, analyze the functions of the variants, and explore the relationships between TSHR genotypes and clinical phenotypes. Methods: In total, 367 patients with CH were recruited for TSHR variant screening using whole-exome sequencing. The effects of the variants were evaluated by in-silico programs such as SIFT and polyphen2. Furthermore, these variants were transfected into 293T cells to detect their Gs/cyclic AMP and Gq/11 signaling activity. Results: Among the 367 patients with CH, 17 TSHR variants, including three novel variants, were identified in 45 patients, and 18 patients carried biallelic TSHR variants. In vitro experiments showed that 10 variants were associated with Gs/cyclic AMP and Gq/11 signaling pathway impairment to varying degrees. Patients with TSHR biallelic variants had lower serum TSH levels and higher free triiodothyronine and thyroxine levels at diagnosis than those with DUOX2 biallelic variants. Conclusions: We found a high frequency of TSHR variants in Chinese patients with CH (12.3%), and 4.9% of cases were caused by TSHR biallelic variants. Ten variants were identified as loss-of-function variants. The data suggest that the clinical phenotype of CH patients caused by TSHR biallelic variants is relatively mild. Our study expands the TSHR variant spectrum and provides further evidence for the elucidation of the genetic etiology of CH.

Myeloid cells interact with a subset of thyrocytes to promote their migration and follicle formation through NF-κB.

The pathogenesis of thyroid dysgenesis (TD) is not well understood. Here, using a combination of single-cell RNA and spatial transcriptome sequencing, we identify a subgroup of NF-κB-activated thyrocytes located at the center of thyroid tissues in postnatal mice, which maintained a partially mesenchymal phenotype. These cells actively protruded out of the thyroid primordium and generated new follicles in zebrafish embryos through continuous tracing. Suppressing NF-κB signaling affected thyrocyte migration and follicle formation, leading to a TD-like phenotype in both mice and zebrafish. Interestingly, during thyroid folliculogenesis, myeloid cells played a crucial role in promoting thyrocyte migration by maintaining close contact and secreting TNF-α. We found that cebpa mutant zebrafish, in which all myeloid cells were depleted, exhibited thyrocyte migration defects. Taken together, our results suggest that myeloid-derived TNF-α-induced NF-κB activation plays a critical role in promoting the migration of vertebrate thyrocytes for follicle generation.

The isl2a transcription factor regulates pituitary development in zebrafish.

Background: ISL LIM homeobox 2, also known as insulin gene enhancer protein ISL-2 (ISL2), is a transcription factor gene that participates in a wide range of developmental events. However, the role of ISL2 in the hypothalamus-pituitary-thyroid axis is largely unknown. In the present study, we characterized the expression patterns of ISL2 and revealed its regulative role during embryogenesis using zebrafish. Methods: We used the CRISPR/Cas9 system to successfully establish homozygous ISL2-orthologue (isl2a and isl2b) knockout zebrafish. Moreover, we utilized these knockout zebrafish to analyze the pituitary and thyroid phenotypes in vivo. For further molecular characterization, in situ hybridization and immunofluorescence were performed. Results: The isl2a mutant zebrafish presented with thyroid hypoplasia, reduced whole-body levels of thyroid hormones, increased early mortality, gender imbalance, and morphological retardation during maturity. Additionally, thyrotropes, a pituitary cell type, was notably decreased during development. Importantly, the transcriptional levels of pituitary-thyroid axis hormones-encoding genes, such as tshba, cga, and tg, were significantly decreased in isl2a mutants. Finally, the thyroid dysplasia in isl2a mutant larvae may be attributed to a reduction in proliferation rather than changes in apoptosis. Conclusions: In summary, isl2a regulates the transcriptional levels of marker genes in hypothalamus-pituitary-thyroid axis, and isl2a knockout causing low thyroid hormone levels in zebrafish. Thus, isl2a identified by the present study, is a novel regulator for pituitary cell differentiation in zebrafish, resulting in thyroid gland hypoplasia and phenotypes of hypothyroidism.

Single-cell analysis of diverse immune phenotypes in malignant pleural effusion.

The complex interactions among different immune cells have important functions in the development of malignant pleural effusion (MPE). Here we perform single-cell RNA sequencing on 62,382 cells from MPE patients induced by non-small cell lung cancer to describe the composition, lineage, and functional states of infiltrating immune cells in MPE. Immune cells in MPE display a number of transcriptional signatures enriched for regulatory T cells, B cells, macrophages, and dendritic cells compared to corresponding counterparts in blood. Helper T, cytotoxic T, regulatory T, and T follicular helper cells express multiple immune checkpoints or costimulatory molecules. Cell-cell interaction analysis identifies regulatory B cells with more interactions with CD4+ T cells compared to CD8+ T cells. Macrophages are transcriptionally heterogeneous and conform to M2 polarization characteristics. In addition, immune cells in MPE show the general up-regulation of glycolytic pathways associated with the hypoxic microenvironment. These findings show a detailed atlas of immune cells in human MPE and enhance the understanding of potential diagnostic and therapeutic targets in advanced non-small cell lung cancer.