TGFβ2 regulates hypothalamic Trh expression through the TGFβ inducible early gene-1 (TIEG1) during fetal development
Graphical Abstract
Introduction
The hypothalamus is a gateway between the endocrine and nervous systems. The thyrotropin-releasing hormone (TRH) is a neuropeptide expressed in various hypothalamic nuclei. The TRH neurons within the hypophysiotropic paraventricular nucleus control thyroid function, but additional effects of TRH on feeding behavior, thermogenesis, and autonomic regulation may be related to other TRH neuron subpopulations (Lechan and Fekete, 2006). Despite the vast knowledge about hypothalamic TRH function, very little is known about the molecular pathways regulating hypothalamic Trh expression during development, and particularly during terminal differentiation, which is characterized by the expression of the phenotype specific neurotransmitter (Hobert et al., 2010). We have previously shown that fetal hypothalamic TRH neurons express transcription factors belonging to the Krüppel-like factor (KLF) family (Guerra-Crespo et al., 2011), and that Klf4 plays an important role in the establishment of the TRH phenotype (Pérez-Monter et al., 2011). Another member of the KLF family enriched in fetal TRH neurons is the transforming growth factor-β (TGFβ) inducible early gene-1 (TIEG1), also called Krüppel-like transcription factor 10 (Klf10) (Guerra-Crespo et al., 2011).
TIEG1 is a member of the three zinc finger family of KLF transcription factors, originally isolated as an early response gene rapidly upregulated after the treatment of human fetal osteoblast cells with TGFβ1 (Subramaniam et al., 1995). TIEG1 acts both as a potent activator or repressor of gene transcription through binding to CACCC- or GC-rich elements within the regulatory region of its target genes (Noti et al, 2004, Ou et al, 2004). The effect of TIEG1 on gene regulation is mediated by its interaction with co-repressor molecules, such as the histone deacetylase mSin3A (Zhang et al., 2001) and the histone demethylase JARID1B (Kim et al., 2010), or with the co-activator histone acetyltransferase p300/CBP-associated factor (PCAF) (Xiong et al., 2012). TIEG1 has a widespread expression pattern (Subramaniam et al., 2005); its expression is detected in various regions of the developing and adult murine brain (Yajima et al., 1997), with TIEG1 mRNA levels peaking a few days after birth in the mouse brain (Jiang et al., 2010). Multiple signaling molecules increase TIEG1 levels in the central nervous system (Consales et al, 2007, Wibrand et al, 2006). TIEG1 is a NGF-responsive immediate early gene during PC12 cell differentiation that induces cell cycle exit without affecting terminal differentiation (Dijkmans et al, 2009, Spittau et al, 2010). In cerebellar granular neuron precursors the BMP2 pathway triggers cell cycle exit, with TIEG1 as a mediator (Alvarez-Rodríguez et al., 2007). Additionally, TIEG1 facilitates apoptosis induced by TGFβ in an oligodendroglial cell line (Bender et al., 2004). However, the role of TIEG1 in neuronal differentiation in vivo, and specifically in the development of hypothalamic phenotypes, is essentially unknown.
TGFβ proteins are multifunctional cytokines that control a wide variety of cellular processes including cell differentiation, proliferation, apoptosis and the specification of developmental fate (Shi and Massague, 2003). Canonical TGFβ signaling is initiated by the binding of a ligand dimer to its serine/threonine kinase receptor at the cell surface. TGFβ ligands (TGFβ1–3) bind the type II TGFβ receptor (TβRII), which causes its recruitment to the type I TGFβ receptor (TβRI). The formation of this complex allows the phosphorylation of the kinase domain of TβRI by TβRII, which in turn triggers cellular changes through transcriptional responses (Shi and Massague, 2003).
During CNS development, TGFβ immunolabeling is most prominent in zones where neuronal differentiation occurs and less intense in zones of active proliferation (Flanders et al., 1991). TGFβ has an anti-mitotic effect on embryonic neural progenitors and increases the expression of neuronal markers in hippocampal and cortical primary cell cultures of developing mouse (Vogel et al., 2010). Similarly, TGFβ2 induces neuronal differentiation during chicken development (Garcia-Campmany and Marti, 2007). TGFβ2-3 induce motoneuron and midbrain dopaminergic neuron survival (Gouin et al, 1996, Krieglstein et al, 1995, Poulsen et al, 1994), and tyrosine hydroxylase expression in the developing chicken and mouse brain (Farkas et al, 2003, Roussa et al, 2008). Interestingly, TGFβ regulates the expression of hypothalamic neuropeptides (POMC, GnRH, AVP) in adult animals (Bouret et al, 2004, Fevre-Montange et al, 2004, Matsumura et al, 2007). The expression pattern of the TGFβ isoforms in different brain areas suggests that they might have phenotype-specific functions. However, the role of TGFβ in the development of the hypothalamus has remained largely unexplored.
Since TGFβ signaling regulates the expression of genes involved in neural development (Estaras et al., 2012), and TIEG1 transcript is a TGFβ induced gene enriched in fetal hypothalamic TRH neurons (Guerra-Crespo et al., 2011), we tested the hypothesis that TGFβ mediated signaling promotes hypothalamic TRH neuronal differentiation. In this report we show that TGFβ2, through TIEG1 action, is part of the differentiation program leading to Trh expression within the hypothalamus.
Section snippets
Animals
Wistar rats were maintained at the institute animal facility in standard environmental conditions (lights on between 07:00 and 19:00 h, temperature 21 ± 2 °C) with rat chow and tap water ad libitum. Animal care and protocols followed the guidelines for the use of animals in neuroscience research of the Society for Neuroscience, USA, and were approved by the Animal Care and Ethics Committee of the Instituto de Biotecnología, UNAM.
Hypothalamic tissue was dissected from Wistar rats at various
TGFβ isoforms and receptors are expressed during hypothalamic development
Since TGFβ signaling plays critical roles in morphogenesis and cell lineage specification during CNS development (Farkas et al, 2003, Krieglstein et al, 2002, Maira et al, 2010, Roussa et al, 2008), and TIEG1, a target of TGFβ signaling, is expressed in the fetal hypothalamus (Guerra-Crespo et al., 2011), we determined the expression profile of the TGFβ isoforms (1, 2 and 3) and TGFβ receptors (TβRI and RII) during rat hypothalamus development. Semi-quantitative RT-PCR analyses revealed that
Discussion
Neurogenesis involves progenitor proliferation and appropriately timed generation of postmitotic neurons. Cell cycle arrest correlates with the appearance of neurite outgrowth, and the synthesis of the phenotype-specific neurotransmitter is determined during the terminal phase of the differentiation. This process is exquisitely regulated by multiple signals including growth factors (FGF, EGF, VEGF), neurotrophins (BDNF, NT3) and transcription factors (reviewed in Pérez-Martínez and Charli, 2006
Acknowledgements
We are grateful to Dr. G. Pedraza-Alva (Instituto de Biotecnología, UNAM) for insightful scientific discussions and critical review of the manuscript, to M. Villa, M. Cisneros, V. Barajas, O. López-Gutiérrez and to Karla F. Meza-Sosa for technical support. We also thank to S. González for maintaining our rat colony. This work was supported by the author's institutions and by grants from DGAPA/UNAM (IN209212), CONACYT/Mexico (155290) and NIH DE 14036.
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Present address: CONACyT, Laboratorio de Investigación en Inmunología y Proteómica. Hospital Infantil de México, Federico Gómez, México D. F.