New regulatory interactions and cellular responses in the isthmic organizer region revealed by alteringGbx2expression

Abstract

The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2–/–; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2–/–; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival.

Sonic hedgehog patterning during cerebellar development

“…The cerebellum arises from a specialized area at the midbrain/hindbrain boundary [72][73][74], where, at embryonic day 8.5 (E8.5), the interaction between homeobox genes Otx2 and Gbx2 defines the Isthmic Organizer region (IO; [75,76]). The IO orchestrates the development of cerebellar and mesencephalic structures through the morphogenic activity of secreted factors, Fgf8 and Wnt1 [77][78][79].…”

A threshold requirement for Gbx2 levels in hindbrain development

“…Previously, Gbx2 has been shown to be essential for the development of r1-r3 and their derivatives (Wassarman et al, 1997). More recently, it has been appreciated that r4-r6, although present, do not display normal gene expression in Gbx2 null homozygotes (Li et al, 2005;Lin et al, 2005). The role of Gbx2 in r3 development is to repress the caudal expansion of Otx2 into this region, as r3 is restored in Otx2…”

Sp8controls the anteroposterior patterning at the midbrain-hindbrain border

“…These findings indicate that beside the expansion of midbrain DA in the rostral hindbrain, it seems that the onset of serotonergic neuron differentiation is at least delayed in mutant embryos. Wurst and Bally-Cuif, 2001;Raible and Brand, 2004;Wassarman et al, 1997;Li and Joyner, 2001;Martinez-Barbera et al, 2001;Li et al, 2002;Li et al, 2005). In the absence of Gbx2 or both Otx2 and Gbx2, the expression domains of these secreted factors exhibit an overlapping disorganized pattern.…”

“…The establishment of a juxtaposed Wnt1 and Fgf8 expression areas at the mes-met border is required for normal function of the IsO (Wassarman et al, 1997;Li and Joyner, 2001;Martinez-Barbera et al, 2001;Li et al, 2002). Otx2 and Gbx2 were shown to promote stable expression and to define the precise positioning of Fgf8 at the MHB (Wassarman et al, 1997;Li and Joyner, 2001;Martinez-Barbera et al, 2001;Li et al, 2002;Li et al, 2005). In the absence of Otx2 and Gbx2, the expression of Sp8 is similar to that reported for Fgf8 and other genes transcribed at the MHB (Martinez-Barbera et al, 2001;Li and Joyner, 2001).…”