BMP signaling through BMPRIA in astrocytes is essential for proper cerebral angiogenesis and formation of the blood–brain-barrier

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Abstract

Bone morphogenetic protein (BMP) signaling is involved in differentiation of neural precursor cells into astrocytes, but its contribution to angiogenesis is not well characterized. This study examines the role of BMP signaling through BMP type IA receptor (BMPRIA) in early neural development using a conditional knockout mouse model, in which Bmpr1a is selectively disrupted in telencephalic neural stem cells. The conditional mutant mice show a significant increase in the number of cerebral blood vessels and the level of vascular endothelial growth factor (VEGF) is significantly upregulated in the mutant astrocytes. The mutant mice also show leakage of immunoglobulin around cerebral microvessels in neonatal mice, suggesting a defect in formation of the blood–brain-barrier. In addition, astrocytic endfeet fail to encircle cortical blood vessels in the mutant mice. These results suggest that BMPRIA signaling in astrocytes regulates the expression of VEGF for proper cerebrovascular angiogenesis and has a role on in the formation of the blood–brain-barrier.

Introduction

Neural stem cells (NSC) are self-renewing, multipotential progenitor cells with the capacity for generating neurons and glial cells including astrocytes and oligodendrocytes. The entire surface of central nervous system is derived from NSCs in the ventricular zone, but actively dividing NSCs generate neurons in all structures of the brain. NSCs in the subventricular zone, which is formed later in embryogenesis, generate neurons, astrocytes and oligodendrocytes (Parnavelas and Nadarajah, 2001, Rao, 1999, Okano, 2002, Lennington et al., 2003). Astrocytes adhere to most of the capillaries in the brain and promote formation of the blood–brain-barrier (BBB).

BMP signaling is critical for gliogenesis (Gross et al., 1996, Mabie et al., 1997, Nakashima et al., 1999a, Reiriz et al., 1999, Lim et al., 2000). For example, in vitro studies demonstrate that astrocytes proliferate actively and extensively branched processes formation in response to exogenous BMPs (Gross et al., 1996, Reiriz et al., 1999, D'Alessandro and Wang, 1994, Jordan et al., 1997, Angley et al., 2003). In addition, overexpression of BMP4 increases the density of astrocytes in multiple brain regions in vivo (Gomes et al., 2003). Several BMP ligands and receptors are expressed in the brain in a cell type-specific and developmental stage-specific manner (Zhang et al., 1998, Mekki-Dauriac et al., 2002), with higher expression during neural development and lower expression in the adult brain. BMPRIA, a type I receptor, is activated primarily by BMP2 and BMP4 (Mishina, 2003), which are expressed in the telencephalic midline (Hebert et al., 2003). BMPRIA stimulates phosphorylation and activation of Smad1, Smad5 and Smad8. Activated Smads form heteromeric complexes and bind directly or indirectly to the promoters of their respective target genes and activate target gene transcription (Derynck et al., 1998). Since disruption of Bmpr1a in mice blocks mesoderm formation and results in intrauterine death before embryonic day 7.5 (E7.5) (Mishina et al., 1995), a conditional allele has been generated to explore its function in neural development (Mishina et al., 2002, Mishina, 2003). These attempts revealed that BMPRIA signaling is critical for dorso-ventral patterning of a developing neural tube (Wine-Lee et al., 2004) and formation of choroid plexus, the most dorsal structure of the neural tube (Hebert et al., 2002). Furthermore, it is recently shown that BMP signaling through BMPRIA is important for cell fate determination in NSCs towards glial lineage (Samanta et al., 2007), and for astrocytic maturation (See et al., 2007). Activation of BMPs signaling is thought to play a role in regulating proliferation, differentiation, and survival on vascular cells (Bobik, 2006 review), but its involvement in regulating endothelial–astrocyte interactions is not yet understood.

Angiogenesis is a multistep process that involves proliferation and migration of endothelial cells (ECs) and generation of extracellular matrix. Angiogenesis is regulated by multiple positive- and negative-regulatory factors, including vascular endothelial growth factor (VEGF), angiopoitins, interferons (alpha/beta/gamma), IL-12, tissue inhibitory metalloproteinase (TIMP)-3 and transforming growth factor-beta 1 (Campbell et al., 1999, Rivieccio et al., 2005, Zeinstra et al., 2006, Constantinescu et al., 2005, Muir et al., 2002, Carmeliet et al., 1996, Petersen et al., 2005, Sidky and Borden, 1987, Raza and Cornelius, 2000, Dhandapani et al., 2005, Dhandapani et al., 2007). VEGF, a prototypical angiogenic growth factor, regulates cell proliferation, vascular permeability, chemotaxis, and survival of ECs and vasculogenesis and angiogenesis in the developing embryo (Ferrara et al., 2003). Astrocytes, whose endfeet encircle neural capillaries, produce angiogenesis modulating factors (Abbott, 2002, Abbott et al., 2006, Lee et al., 2003), and together with ECs, play a key role in forming and maintaining the blood–brain-barrier (BBB). It has been suggested that closure of BBB is correlated to the maturation of astrocytes (DeBault and Cancilla, 1980, Janzer and Raff, 1987). VEGF and BMPs also modulate functions of peripheral ECs (Hoeben et al., 2004, Holmes and Zachary, 2005, Valdimarsdottir et al., 2002, Itoh et al., 2004).

In this study, we conditionally disrupted Bmpr1a in a neural stem cell-specific manner to examine the functional roles of Bmpr1a during neural development (Mishina et al., 2002, Yuhki et al., 2004). Our data demonstrated that BMPRIA was essential in astrocytes for down-regulation of VEGF expression during cerebral vascular angiogenesis. It was also demonstrated that BMPRIA in astrocytes played a critical role for interactions between ECs and astrocytes to form a functional BBB.

Section snippets

BMPRIA signaling is dispensable for forming the cortical layer

Emx1Cre/+ mice (Iwasato et al., 2000) were crossed to CAG-CAT-LacZ reporter mice to obtain double heterozygous (Emx1-Cre/LacZ) mice; these animals were used to confirm the tissue- and stage-specificity of expression of the Emx1-Cre transgene. At postnatal day 14 (P14), Emx1-Cre/LacZ mice expressed Cre recombinase [as evidenced by the staining for β-galactosidase (β-Gal) activity] in the dorsal telencephalon and in > 90% of astrocytes in the marginal zone (MZ) (Fig. 1B). Cre recombinase was also

Discussion

Previous studies demonstrate that BMP signaling plays significant roles in patterning of the medial–lateral axis during early neural development (Furuta et al., 1997, Hebert et al., 2002) and in gliogenesis during late neural development (Gross et al., 1996, Mabie et al., 1997, Nakashima et al., 1999a, Reiriz et al., 1999, Lim et al., 2000, Gomes et al., 2003). This study provides evidences that BMP signaling through BMPRIA regulates cerebrovascular angiogenesis, modulates interactions between

Generation of mouse lines

For evaluation of the Cre activities, Emx1Cre/+ mice were bred with CAG-CAT-Z reporter mice (Sakai and Miyazaki, 1997), and double heterozygous (Emx1-Cre/LacZ) mice were obtained. We crossed Emx1Cre/+ mice with CMV-Z/AP reporter mice (Lobe et al., 1999), and obtained double heterozygous (Emx1-Cre/AP+) mice. Emx1-Cre mice and floxed-Bmpr1a mice were maintained as previously described (Yuhki et al., 2004). The present study was carried out according to the Guide for the Care and Use of laboratory

Acknowledgments

We thank Drs. J. Harry, M. Ray, H. Hama, M. Yuhki, and M. Ogawa, for their kind advice. We thank N. Kitamura, H. Kishida, and Y. Sakamaki (RIKEN BSI-RRC) for their expert technical assistance. The CAG-CAT-LacZ reporter mice were provided by Dr. J. Miyazaki. This work was supported by MEXT Grant-in-Aid for Young Scientists (B) and #16047231 to M.Y. and by the Intramural Research Program of the NIEHS, NIH (ES071003-10) to Y. M.

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