Effects of Wnt1 signaling on proliferation in the developing mid-/hindbrain region

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Abstract

The secreted glycoprotein WNT1 is expressed in the caudal midbrain and is essential for proper development of the entire mid-/hindbrain region. To get better insights into Wnt1 function in the mid-/hindbrain region, we ectopically expressed Wnt1 under the control of the endogenous En1 promoter, thereby extending Wnt1 expression rostrally into the anterior midbrain and caudally into rhombomere 1. In these transgenic mice, the position of the mid-/hindbrain organizer is not altered and pattern formation is not changed. During midgestation, ectopic Wnt1 induced strong overproliferation of precursor cells only in the caudal midbrain in a gene dosage-dependent manner. Enhanced proliferation is at least in part mediated by shortening of the cell cycle length. In adults, Wnt1 exhibited a cell size promoting effect specifically on neurons. We suggest that Wnt1 acts as a regulator of proliferation of specific precursor populations in the developing mid-/hindbrain region and is only secondarily involved in maintenance of the mid-/hindbrain organizer.

Introduction

Local organizing centers within the central nervous system (CNS) emit inductive factors controlling patterning of adjacent regions. One of the best-characterized organizing centers within the CNS is the mid-/hindbrain organizer (MHO), which is located at the boundary between the developing mid- and hindbrain (for reviews, see Joyner et al., 2000, Rhinn and Brand, 2001, Wurst and Bally-Cuif, 2001). In chicken, transplantation of the MHO into the diencephalon induces the formation of ectopic midbrain and cerebellar tissue. Transplantation of the MHO into the caudal hindbrain induces the formation of ectopic cerebellar tissue Bally-Cuif and Wassef, 1994, Martinez et al., 1991, Martinez et al., 1995. These experiments clearly demonstrate that this brain region is capable of organizing the development of adjacent territories. The position of the MHO is determined by the juxtaposition and interaction of the homeodomain transcription factors OTX2 and GBX2 Broccoli et al., 1999, Garda et al., 2001, Katahira et al., 2000, Millet et al., 1999, Tour et al., 2002. In a mouse model, in which Otx2 is expressed under the control of the endogenous En1 promotor, Otx2 expression is extended caudally across the mid-/hindbrain boundary (MHB) into rhombomere 1. In these animals, a caudal shift of the expression of MHO marker genes including Wnt1 could be observed. As a consequence, gross morphological alterations such as an enlargement of the dorsal midbrain and reduction of the vermis were observed (Broccoli et al., 1999). Likewise, a rostral shift of the Gbx2 expression domain led to a rostral shift of the MHO (Millet et al., 1999).

Gene inactivation experiments have shown that the homeodomain proteins EN1, EN2, and the paired domain factor PAX2, which are expressed across the MHB are necessary for development of the mid-/hindbrain region Joyner et al., 1991, Urbanek et al., 1997, Wurst et al., 1994. Likewise, FGF8, its receptor FGFR-1, and WNT1 are essential for the development of the entire mid-/hindbrain region Chi et al., 2003, McMahon and Bradley, 1990, McMahon et al., 1992, Meyers et al., 1998, Thomas and Capecchi, 1990, Trokovic et al., 2003. MHO activity can partially be mimicked by FGF8 when released from acrylic beads transplanted into the forebrain or hindbrain Crossley et al., 1996, Liu and Joyner, 2001, Liu et al., 1999, Martinez et al., 1999, Shamim et al., 1999. In tissue surrounding the FGF8 bead, expression of mid-/hindbrain marker genes is ectopically induced and mid-/hindbrain-like structures subsequently develop around the bead (Martinez et al., 1999). Thus, Fgf8 is a key mediator of MHO activity.

Much less is known about the precise function of Wnt1. During embryonic development, Wnt1 is first expressed at E8.0 in a broad band in the prospective midbrain region Bally-Cuif and Wassef, 1994, Davis and Joyner, 1988, Parr et al., 1993, Wilkinson et al., 1987. By E9.5, the Wnt1 expression domain has narrowed to a thin ring at the caudal midbrain bordering the hindbrain. It is also detected in narrow parasagittal stripes in the ventral mesencephalic flexure, the dorsal midline of the caudal forebrain, midbrain, caudal hindbrain, and the spinal cord Bally-Cuif and Wassef, 1994, Parr et al., 1993, Wilkinson et al., 1987. Inactivation of the Wnt1 gene resulted in a deletion of the entire mid-/hindbrain region concomitant with a stepwise loss of En1 and Fgf8 expression Lee et al., 1997, McMahon and Bradley, 1990, McMahon et al., 1992, Thomas and Capecchi, 1990. These results clearly show that Wnt1 function is necessary for the development of the entire mid-/hindbrain region. However, the exact nature of this function remains to be elucidated.

During development and in adulthood, WNT proteins have a variety of tissue-specific functions, most of which could contribute to the loss of function phenotype. WNT functions include control of tissue patterning, cell fate determination, cell migration, cell polarity, apoptosis, and proliferation (for reviews, see Miller, 2002, Polakis, 2000).

To distinguish between the different modes of action of Wnt1 specifically during mouse mid-/hindbrain development and in respect to its role in MHO function, we ectopically expressed it under the control of the En1 promotor using a knock-in approach (En1+/Wnt1). This strategy resulted in an ectopic and overexpression of Wnt1 in the midbrain and ectopic expression in the rostral hindbrain from E8.5 of embryonic development until adulthood. This transgenic model has the advantage of ectopically expressing Wnt1 in a suitable spatiotemporal manner for studying its function specifically during mid-/hindbrain development.

While in these transgenic animals, the patterning activity of the MHO remains largely unaltered, the dorsocaudal midbrain is dramatically enlarged in size in a Wnt1 dosage-dependent manner. Here, the term “patterning” will be used in respect to development and maintenance of regional identity. Therefore, in this strict sense, tissue growth is not regarded as a patterning effect. We demonstrate that Wnt1 misexpression enhances proliferation of neural progenitor cells in the dorsal midbrain and that shortening the cell cycle of proliferating cells contributes to this effect. We further show that ectopic Wnt1 in the adult caudal midbrain promotes an increase in size of neurons. These experiments indicate that in contrast to Fgf8, Wnt1 is not a key mediator of MHO patterning activity. Instead, in the developing mid-/hindbrain region, Wnt1 controls proliferation of specific progenitor cell populations via the canonical Wnt/β-catenin pathway.

Section snippets

Ectopic Wnt1 expression and activation of target genes in vivo

To determine the function of Wnt1 during mid-/hindbrain development, we extended the expression domain of Wnt1 ectopically across the MHB during and after the formation of the mid-/hindbrain region. For this purpose, we replaced the coding sequence of En1 with a Wnt1 cDNA (followed by an IRES lacZ gene cassette to facilitate monitoring ectopic expression) using a gene knock-in approach in embryonic stem cells. As a result, Wnt1 and lacZ are now under control of the regulatory elements of En1

Conclusions

Our results suggest that one of the major functions of Wnt1 in the mid-/hindbrain region is to regulate cell proliferation of progenitor cells in a strict spatiotemporal manner and secondarily to maintain the MHO. Furthermore, the shortening of the cell cycle length during development and the increase of cell size in adult transgenic animals imply that enhancing cellular growth is contributing to the proliferation enhancing effect of Wnt1 on neural progenitor cells. Further experiments will

Knock-in targeting strategy

A mouse Wnt1 cDNA fragment (1879 bp, SalI fragment of vector pBSKS + Wnt1 kindly provided by M. Wassef) was cloned blunt into the BamHI site of the IRES-lacZ vector (provided by A. Mallamaci). The 5.57 kb BglII/SalI Wnt1IRESlacZ fragment was cloned blunt into the KpnI site of the En1 3′ shuttle vector (Hanks et al., 1995). The construct was electroporated into R1 ES cells, which are derived from a mixed 129SV background. Homologous recombination events were detected by genotyping using HindIII

Acknowledgements

We thank Drs. Laure Bally-Cuif, Jordi Guimera, Gail Martin, Nilima Prakash, and Martin Raff for critical scientific input; Rudolf Grosschedl, Peter Gruss, Alexandra L. Joyner, Antonello Mallamaci, Gail Martin, Salvador Martinez, Charles J. Sherr, Antonio Simeone, and Marion Wassef for plasmids and probes; Luise Jennen for electron microscopic assistance; Annerose Kurz-Drexler, Susanne Bourier, Stefanie Pirrung, Sava Michailidou for excellent technical assistance; and Resi Wandrowetz, Rosi

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