During development of the central nervous system, neural cells respond to several external cues that influence cell proliferation, differentiation, axonal growth and synaptogenesis. Thyroid hormone plays a critical role in each of these processes. Previously, we showed that Krüppel-like factor 9 (KLF9), a zinc finger transcription factor, is strongly and directly induced by liganded thyroid hormone receptors, and it mediates the actions of thyroid hormone on neuronal differentiation during late fetal development. Here we analyzed the molecular mechanisms by which KLF9 maintains neuronal structure, and inhibits regeneration in juvenile and adult neuronal cells. We also investigated the actions of the closely related transcription factor KLF13, which is paralogous to KLF9. We engineered the adult mouse hippocampus-derived cell line HT22 to control Klf9 or Klf13 expression by addition of doxycycline. We also used CRISPR/Cas9 genome editing to generate Klf9 or Klf13 knock out (KO), and Klf9+Klf13 double KO HT22 cell lines. To induce neurite outgrowth, we treated cells with forskolin (FK)+IBMX, which increases intracellular cAMP; elevated cAMP is a hallmark of regenerative responses of neurons to injury. Our results show that FK+IBMX increased neurite length in the parent HT22 cell line, and this action was enhanced in Klf9 and Klf13 single KO cells, and was even greater in double KO cells. By contrast, the stimulatory effect of FK+IBMX on neurite outgrowth was blocked by simultaneous forced expression of Klf9 or Klf13 in parent HT22 cells. This effect on neurite outgrowth was confirmed in primary mouse hippocampal neurons, where electroporation of expression plasmids for Klf9 or Klf13 suppressed FK+IBMX-induced neurite extension compared with empty vector-transfected cells. Analysis of RNA-seq data obtained from HT22 cells following 8 hr of induced Klf9 or Klf13 expression showed that both proteins impact the cAMP signaling pathway. Using transfection-reporter assays and chromatin immunoprecipitation, we confirmed that several genes in this pathway are direct targets of both KLFs. Our findings suggest that KLF9 and KLF13 may cooperate to maintain the differentiated state of mammalian neurons and thereby block regeneration, in part, by repressing the cAMP signaling pathway.

Kruppel-Like Factors 9 and 13 Cooperate to Maintain Mammalian Neuronal Differentiation

We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear.Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

The many faces of nuclear receptor biology

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Kruppel-Like Factors 9 and 13 Cooperate to Maintain Mammalian Neuronal Differentiation

Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

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