dissection-coronal-slice-preparation-developing-mouse-pituitary

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Second Military Medical University

The alpha-fetoprotein (AFP) gene is highly activated in fetal liver but is dramatically repressed shortly after birth. The mechanisms that underlie AFP transcriptional repression in postpartum liver are not well understood. AFP enhancer, repressor region, and promoter are implicated to be involved in AFP postnatal repression, but the major transcriptional repressor remains undefined. We previously identified a zinc finger protein gene ZBTB20. To determine its physiological functions in vivo, we have generated hepatocyte-specific ZBTB20 knockout mice by the Cre/loxP approach and demonstrated here that ZBTB20 ablation in liver led to dramatic derepression of the AFP gene in entire liver throughout adult life, although the hepatocytes were normally under nonproliferating status. Furthermore, we found that ZBTB20 was a transcriptional repressor capable of specifically inhibiting AFP promoter-driven transcriptional activity. Liver chromatin immunoprecipitation and mobility shift assays showed that ZBTB20 bound to AFP promoter directly. ZBTB20 was developmentally activated in liver after birth and inversely correlated with its AFP gene expression, suggesting that activated ZBTB20 expression in liver mediated AFP gene repression. Our data point to ZBTB20 as a key regulator governing AFP gene transcription and postulate a new model for the postnatal gene repression of AFP in liver.

Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver.

Zbtb20 is a member of the POK family of proteins, which function primarily as transcriptional repressors via interactions mediated by their conserved C(2)H(2) Krüppel type zinc finger and BTB/POZ domains. To define the function of Zbtb20 in vivo, we generated knockout mice by homologous recombination. Zbtb20 null mice display a stark phenotype characterized by postnatal growth retardation, metabolic dysfunction, and lethality. Zbtb20 knockout mice displayed abnormal glucose homeostasis, hormonal responses, and depletion of energy stores, consistent with an energetic deficit. Additionally, increased serum bilirubin and alanine aminotransferase levels were suggestive of liver dysfunction. To identify potential liver-specific Zbtb20 target genes, we performed transcript profiling studies on liver tissue from Zbtb20 knockout mice and wild-type littermate controls. These studies identified sets of genes involved in growth, metabolism, and detoxification that were differentially regulated in Zbtb20 knockout liver. Transgenic mice expressing Zbtb20 in the liver were generated and crossed onto the Zbtb20 knockout background, which resulted in no significant normalization of growth or glucose metabolism but a significant increase in life span compared to controls. These data indicate that the phenotype of Zbtb20 knockout mice results from liver-dependent and -independent defects, suggesting that Zbtb20 plays nonredundant roles in multiple organ systems.

Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis.

The development of hippocampal circuitry depends on the proper assembly of correctly specified and fully differentiated hippocampal neurons. Little is known about factors that control the hippocampal specification. Here, we show that zinc finger protein Zbtb20 is essential for the specification of hippocampal CA1 field identity. We found that Zbtb20 expression was initially activated in the hippocampal anlage at the onset of corticogenesis, and persisted in immature hippocampal neurons. Targeted deletion of Zbtb20 in mice did not compromise the progenitor proliferation in the hippocampal and adjacent transitional ventricular zone, but led to the transformation of the hippocampal CA1 field into a transitional neocortex-like structure, as evidenced by cytoarchitectural, neuronal migration, and gene expression phenotypes. Correspondingly, the subiculum was ectopically located adjacent to the CA3 in mutant. Although the field identities of the mutant CA3 and dentate gyrus (DG) were largely maintained, their projections were severely impaired. The hippocampus of Zbtb20 null mice was reduced in size, and exhibited increased apoptotic cell death during postnatal development. Our data establish an essential role of Zbtb20 in the specification of CA1 field identity by repressing adjacent transitional neocortex-specific fate determination.

Zbtb20 is essential for the specification of CA1 field identity in the developing hippocampus.

Pancreatic β-cells can precisely sense glucose stimulation and accordingly adjust their insulin secretion. Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme, but its physiological significance in β-cells is not established. Here we determined its physiological role in regulating glucose sensing and insulin secretion of β-cells. Considerable FBPase mRNA was detected in normal mouse islets and β-cell lines, although their protein levels appeared to be quite low. Down-regulation of FBP1 in MIN6 cells by small interfering RNA could enhance the glucose-stimulated insulin secretion (GSIS), whereas FBP1-overexpressing MIN6 cells exhibited decreased GSIS. Inhibition of FBPase activity in islet β-cells by its specific inhibitor MB05032 led to significant increase of their glucose utilization and cellular ATP to ADP ratios and consequently enhanced GSIS in vitro. Pretreatment of mice with the MB05032 prodrug MB06322 could potentiate GSIS in vivo and improve their glucose tolerance. Therefore, FBPase plays an important role in regulating glucose sensing and insulin secretion of β-cells and serves a promising target for diabetes treatment.

Fructose-1,6-bisphosphatase regulates glucose-stimulated insulin secretion of mouse pancreatic beta-cells.

Fructose-1,6-bisphosphatase (FBP)-1 is a gluconeogenic enzyme that regulates glucose metabolism and insulin secretion in β cells, but little is known about how its transcription is controlled. The zinc finger protein ZBTB20 regulates glucose homeostasis, so we investigated its effects on expression of FBP-1.

The zinc finger protein ZBTB20 regulates transcription of fructose-1,6-bisphosphatase 1 and β cell function in mice.

The mammalian hippocampus harbours neural circuitry that is crucial for associative learning and memory. The mechanisms that underlie the development and regulation of this complex circuitry are not fully understood. Our previous study established an essential role for the zinc finger protein Zbtb20 in the specification of CA1 field identity in the developing hippocampus. Here, we show that conditionally deleting Zbtb20 specifically in mature CA1 pyramidal neurons impaired hippocampus-dependent memory formation, without affecting hippocampal architecture or the survival, identity and basal excitatory synaptic activity of CA1 pyramidal neurons. We demonstrate that mature CA1-specific Zbtb20 knockout mice exhibited reductions in long-term potentiation (LTP) and NMDA receptor (NMDAR)-mediated excitatory post-synaptic currents. Furthermore, we show that activity-induced phosphorylation of ERK and CREB is impaired in the hippocampal CA1 of Zbtb20 mutant mice. Collectively, these results indicate that Zbtb20 in mature CA1 plays an important role in LTP and memory by regulating NMDAR activity, and activation of ERK and CREB.

Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons.

Adaptor protein, phosphotyrosine interaction, pleckstrin homology domain and leucine zipper containing 1 (APPL1) is an adapter protein that positively mediates adiponectin signalling. Deficiency of APPL1 in the target tissues of insulin induces insulin resistance. We therefore aimed, in the present study, to determine its role in regulating pancreatic beta cell function.

Deficiency of APPL1 in mice impairs glucose-stimulated insulin secretion through inhibition of pancreatic beta cell mitochondrial function.

In mammals, pain sensation is initiated by the detection of noxious stimuli through specialized transduction ion channels and receptors in nociceptive sensory neurons. Transient receptor potential (TRP) channels are the key sensory transducers that confer nociceptors distinct sensory modalities. However, the regulatory mechanisms about their expression are poorly defined. Here we show that the zinc-finger protein ZBTB20 regulates TRP channels expression in nociceptors. ZBTB20 is highly expressed in nociceptive sensory neurons of dorsal root ganglia. Disruption of ZBTB20 in nociceptors led to a marked decrease in the expression levels of TRPV1, TRPA1 and TRPM8 and the response of calcium flux and whole-cell currents evoked by their respective specific agonists. Phenotypically, the mice lacking ZBTB20 specifically in nociceptors showed a defect in nociception and pain sensation in response to thermal, mechanical and inflammatory stimulation. Our findings point to ZBTB20 as a critical regulator of nociception and pain sensation by modulating TRP channels expression in nociceptors.

ZBTB20 regulates nociception and pain sensation by modulating TRP channel expression in nociceptive sensory neurons.

The terminal differentiation of hypertrophic chondrocytes is a tightly regulated process that plays a pivotal role in endochondral ossification. As a negative regulator, Sox9 is essentially downregulated in terminally differentiated hypertrophic chondrocytes. However, the underlying mechanism of Sox9 silencing is undefined. Here we show that the zinc finger protein Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes by repressing Sox9. In the developing skeleton of the mouse, Zbtb20 protein is highly expressed by hypertrophic chondrocytes from late embryonic stages. To determine its physiological role in endochondral ossification, we have generated chondrocyte-specific Zbtb20 knockout mice and demonstrate that disruption of Zbtb20 in chondrocytes results in delayed endochondral ossification and postnatal growth retardation. Zbtb20 deficiency caused a delay in cartilage vascularization and an expansion of the hypertrophic zone owing to reduced expression of Vegfa in the hypertrophic zone. Interestingly, Sox9, a direct suppressor of Vegfa expression, was ectopically upregulated at both mRNA and protein levels in the late Zbtb20-deficient hypertrophic zone. Furthermore, knockdown of Sox9 greatly increased Vegfa expression in Zbtb20-deficient hypertrophic chondrocytes. Our findings point to Zbtb20 as a crucial regulator governing the terminal differentiation of hypertrophic chondrocytes at least partially through repression of Sox9.

Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9.

Alpha-fetoprotein (AFP) represents a classical model system to study developmental gene regulation in mammalian cells. We previously reported that liver ZBTB20 is developmentally regulated and plays a central role in AFP postnatal repression. Here we show that ZBTB20 is a sequence-specific transcriptional repressor of AFP. By ELISA-based DNA-protein binding assay and conventional gel shift assay, we successfully identified a ZBTB20-binding site at -104/-86 of mouse AFP gene, flanked by two HNF1 sites and two C/EBP sites in the proximal promoter. Importantly, mutation of the core sequence in this site fully abolished its binding to ZBTB20 in vitro, as well as the repression of AFP promoter activity by ZBTB20. The unique ZBTB20 site was highly conserved in rat and human AFP genes, but absent in albumin genes. These help to explain the autonomous regulation of albumin and AFP genes in the liver after birth. Furthermore, we demonstrated that transcriptional repression of AFP gene by ZBTB20 was liver-specific. ZBTB20 was dispensable for AFP silencing in other tissues outside liver. Our data define a cognate ZBTB20 site in AFP promoter which mediates the postnatal repression of AFP gene in the liver.

ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene.

The recombination efficiency and cell specificity of Cre driver lines are critical for exploring pancreatic β cell biology with the Cre/LoxP approach. Some commonly used Cre lines are based on the short Ins2 promoter fragment and show recombination activity in hypothalamic neurons; however, whether this stems from endogenous Ins2 promoter activity remains controversial. In this study, we generated Ins2-Cre knockin mice with a targeted insertion of IRES-Cre at the Ins2 locus and demonstrated with a cell lineage tracing study that the Ins2 gene is not transcriptionally active in the hypothalamus. The Ins2-Cre driver line displayed robust Cre expression and activity in pancreatic β cells without significant alterations in insulin expression. In the brain, Cre activity was mainly restricted to the choroid plexus, without significant recombination detected in the hippocampus or hypothalamus by the LacZ or fluorescent tdTomato reporters. Furthermore, Ins2-Cre mice exhibited normal glucose tolerance and insulin secretion upon glucose stimulation in vivo. In conclusion, this Ins2-Cre driver line allowed high-fidelity detection of endogenous Ins2 promoter activity in vivo, and the negative activity in the hypothalamus demonstrated that this system is a promising alternative tool for studying β cell biology.

Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons.

The anterior pituitary harbours five distinct hormone-producing cell types, and their cellular differentiation is a highly regulated and coordinated process. Here we show that ZBTB20 is essential for anterior pituitary development and lactotrope specification in mice. In anterior pituitary, ZBTB20 is highly expressed by all the mature endocrine cell types, and to some less extent by somatolactotropes, the precursors of prolactin (PRL)-producing lactotropes. Disruption of Zbtb20 leads to anterior pituitary hypoplasia, hypopituitary dwarfism and a complete loss of mature lactotropes. In ZBTB20-null mice, although lactotrope lineage commitment is normally initiated, somatolactotropes exhibit profound defects in lineage specification and expansion. Furthermore, endogenous ZBTB20 protein binds to Prl promoter, and its knockdown decreases PRL expression and secretion in a lactotrope cell line MMQ. In addition, ZBTB20 overexpression enhances the transcriptional activity of Prl promoter in vitro. In conclusion, our findings point to ZBTB20 as a critical regulator of anterior pituitary development and lactotrope specification.

ZBTB20 is required for anterior pituitary development and lactotrope specification.

Hepatic de novo lipogenesis (DNL) converts carbohydrates into triglycerides and is known to influence systemic lipid homoeostasis. Here, we demonstrate that the zinc finger protein Zbtb20 is required for DNL. Mice lacking Zbtb20 in the liver exhibit hypolipidemia and reduced levels of liver triglycerides, along with impaired hepatic lipogenesis. The expression of genes involved in glycolysis and DNL, including that of two ChREBP isoforms, is decreased in livers of knockout mice. Zbtb20 binds to and enhances the activity of the ChREBP-α promoter, suggesting that altered metabolic gene expression is mainly driven by ChREBP. In addition, ChREBP-β overexpression largely restores hepatic expression of genes involved in glucose and lipid metabolism, and increases plasma and liver triglyceride levels in knockout mice. Finally, we show that Zbtb20 ablation protects from diet-induced liver steatosis and improves hepatic insulin resistance. We suggest ZBTB20 is an essential regulator of hepatic lipogenesis and may be a therapeutic target for the treatment of fatty liver disease.

Article	Year
Zinc finger protein ZBTB20 is a key repressor of alpha-fetoprotein gene transcription in liver. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 18669658	2008
Zinc finger protein Zbtb20 is essential for postnatal survival and glucose homeostasis. Molecular and cellular biology\| PubMed ID: 19273596	2009
Zbtb20 is essential for the specification of CA1 field identity in the developing hippocampus. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 20308569	2010
Fructose-1,6-bisphosphatase regulates glucose-stimulated insulin secretion of mouse pancreatic beta-cells. Endocrinology\| PubMed ID: 20719858	2010
The zinc finger protein ZBTB20 regulates transcription of fructose-1,6-bisphosphatase 1 and β cell function in mice. Gastroenterology\| PubMed ID: 22374165	2012
Regulation of hippocampus-dependent memory by the zinc finger protein Zbtb20 in mature CA1 neurons. The Journal of physiology\| PubMed ID: 22777671	2012
Deficiency of APPL1 in mice impairs glucose-stimulated insulin secretion through inhibition of pancreatic beta cell mitochondrial function. Diabetologia\| PubMed ID: 23793716	2013
ZBTB20 regulates nociception and pain sensation by modulating TRP channel expression in nociceptive sensory neurons. Nature communications\| PubMed ID: 25369838	2014
Zbtb20 regulates the terminal differentiation of hypertrophic chondrocytes via repression of Sox9. Development (Cambridge, England)\| PubMed ID: 25564625	2015
ZBTB20 is a sequence-specific transcriptional repressor of alpha-fetoprotein gene. Scientific reports\| PubMed ID: 26173901	2015
Knockin of Cre Gene at Ins2 Locus Reveals No Cre Activity in Mouse Hypothalamic Neurons. Scientific reports\| PubMed ID: 26830324	2016
ZBTB20 is required for anterior pituitary development and lactotrope specification. Nature communications\| PubMed ID: 27079169	2016
Regulation of hepatic lipogenesis by the zinc finger protein Zbtb20. Nature communications\| PubMed ID: 28327662	2017

Zhifang Xie

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