THE JOURNAL OF COMPARATIVE NEUROLOGY 374:246-258 (1996) Early Deletion of Neuromeres in Wnt-l- / - Mutant Mice: Evaluation by Morphological and Molecular Markers

The Wnt-1 gene is required for the development of midbrain and cerebellum; previous work showed that knockout of Wnt-l causes the loss of most molecular markers of these structures in early embryos and deletion of these structures by birth. However, neither the extent of early neuronal defects nor any possible alterations in structures adjacent to presumptive midbrain and cerebellum were examined. By using a neuron-specific antibody and fluorescent axon tracers, we show that central and peripheral neuronal development are altered in mutants during initial axonogenesis on embryonic day 9.5. The absence of neuronal landmarks, including oculomotor and trochlear nerves and cerebellar plate, suggests that both mesencephalon and rhombomere 1 (rl) are deleted, with the remaining neural tube fused to form a new border between the caudalmost portion of the prosencephalon (prosomere 1, or p l ) and r2. Central axons accurately traverse this novel border by forming normal longitudinal tracts into the rhombencephalon, implying that the cues that direct these axom are aligned across neuromeres and are not affected by the deletion. The presence of intact p l and r2 is further supported by the retention of markers for these two neuromeres, including a marker of p l , the Sim-2 gene, and an r2-specific lacZ transgene in mutant embryos. In addition, alterations in the Sim-2 expression domain in ventral prosencephalon, rostral to p l , provide novel evidence for Wnt-1 function in this region. Indexing terms: axon guidance, mesencephalon, cranial nerves, rhombencephalon, forebrain o 1996 Wiley-Liss, Inc. The first regional differentiation in the brain is surely controlled genetically, and many candidate regulatory factors are expressed early in regionally restricted patterns. Of the three primary vesicles, prosencephalon, mesencephalon, and rhombencephalon, most work has focused on the latter, where nested expression patterns of homeobox transcription factors are thought to specify rhombomeric identity (Wilkinson and Krumlauf, 1990; Hunt et al., 1991). A large number of genes have also been shown to be expressed early in the more rostral neural tube (reviewed in Puelles and Rubenstein, 19931, but few of these genes have been tested for functional roles in brain development. One of the best characterized of these candidates is the Wnt-1 gene, a member of a family of related genes that are expressed early in vertebrate embryogenesis (McMahon, 1992; Nusse and Varmus, 1992). Like its Drosophila homologue, wingless (wg), the mouse gene Wnt-1 encodes a secreted glycoprotein that may play a role in directing cell fates. On embryonic day (E) 8.0, this gene is transcribed in a restricted region of the neural plate, probably the presumptive mesencephalon excluding the ventral midline, and as the neural tube forms, the expression pattern changes dynamically (Wilkinson et al., 1987; Parr et al., 1993). By E9.0-E9.5, expression in the brain becomes restricted to a narrow circumferential ring just rostral to the border between the mesencephalon and the rhombencephalon, and two strips, one dorsal and one ventral, both of which extend rostrally through the mesencephalon into the prosencephalon. Expression is absent in rhombomere 1 (rl) , but a dorsal strip of expression initiating in r2 extends caudally through the rhombencephalon and the spinal cord. Accepted May 23,1996 Address reprint requests to Grant Mastick, Department of Biology, University of Michigan, Ann Arbor, MI 48109. E-mail: grants@ umich.edu o 1996 WILEY-LISS, INC. Wnt-1EMBRYONIC BRAIN 247 Fig. 1. Neuromeric subdivisions of the neural tube on E9.5. A: Wildtype embryo. The neuromeres (prosomeres 1 and 2, mesencephalon, and rhombomeres 1 and 2: pl , p2, m, r l , and r2, respectively) are separated by interneuromeric constrictions indicated by short lines at the dorsal surface. An additional boundary marker is the thin triangular roof (*) that lies just caudal to the rl-r2 border. The cephalic flexure (cD is the ventrally directed hairpin turn of the neural tube. The front of the neural tube, marked roughly by the olfactory placode (010 in the overlying skin, is rotated nearly 180” relative to the rhombencephalon. Wnt-1 mutants generated by gene targeting result in embryos with large deletions in the brain. The boundaries of these deletions were estimated partly from morphology but mostly from molecular markers (immunoreactivity or in situ hybridization) on the assumption that the absence of a marker implied the loss of the subdivision in which the marker normally appeared. On E9.5, the brain of Wnt-1-1embryos is smaller than that of the wildtype (McMahon et al., 19921, principally because the mesencephalon (recognized as the most sharply curved part of the dorsal surface of the brain) is smaller in the mutant, as a comparison of Figure 1A and 1B shows. In addition to the change in morphology, the expression patterns of other Wnt genes are altered on E9.5; Wnt-7b expression in caudal dorsal mesencephalon is lost, and a Wnt-5u expression domain, normally extending from the ventral forebrain into the ventral mesencephalon, is reduced in size, suggesting that part of the mesencephalon must be missing. This conclusion is supported by the loss of engruiled (En) expression in the mutant (McMahon et al., 1992). Both En-1 and En-2 are expressed initially in both the mesencephalon and r l , but expression later disappears at stages when it persists in wildtype. These observations led McMahon et al. (1992) to estimate that “much of the midbrain and some of the metencephalon are deleted in the Wnt-1-1embryo” (p. 582). Many days later in development, the adult brain structure can be assessed with histological sections and a comparison with the normal neuroanatomy. By these criteria, homozygous Wnt-2-1mice lack the cerebellum and most or all of the midbrain on E14.5 and E16.5 (McMahon and Bradley, 1990; but see Thomas and Capecchi, 1990). The midbrain is derived from the mesencephalon and the cerebellum from the mesencephalon and r l (Martinez and Alvarado-Mallart, 19891, so the late embryonic deficits correlate well with those noted earlier. But the issue of whether entire early subdivisions (e.g., mesencephalon The cerebral vesicle (cv), optic vesicle (op), and trigeminal ganglion (gV) are shown. B: Wnt-1-lembryo, littermate of A. The p2-pl constriction is evident, but the pl-m and m-rl constrictions are not. The rostra1 border of r2 is evident from the thin roof (*I. The trigeminal ganglion was removed to reveal the very narrow cephalic flexure (cD. The front of the neural tube is rotated about 45” less than in the wildtype, which is consistent with a deletion of a wedge, wide side dorsal, including most or all of mesencephalon and r l . andior rl) were deleted requires examination of the early brain with independent markers that can reveal the neural organization at that time. Current understanding of the organization of the early brain is based on neuromeres and the initial patterns of neurons, which are evident at E9.5, when the Wnt-1-1embryo shows the first defects. Soon after neural tube closure, the primary vesicles are subdivided into neuromeres, a series of annular subdivisions marked by transverse bulges, and separated from one another by constrictions in the wall of the neural tube (reviewed in Puelles et al., 1987). The neuromeres have regional properties that are thought to be important developmentally, including rate of proliferation (Bergquist and Kallen, 1954; Guthrie et al., 19911, patterns of gene expression (Bulfone et al., 1993; Puelles and Rubenstein, 1993) and of neurogenesis (Puelles et al., 1987; Lumsden and Keynes, 1989), and restriction of cellular migration (Fraser et al., 1990; Figdor and Stern, 1993). The number of neuromeres increases steadily with development, but we are concerned primarily with the rhombomeres (rl-r7), the mesencephalon (mes; which is not apparently subdivided at this time), and the two most caudal subunits in the prosencephalon, prosomeres 1 and 2 (pl and p2; see Fig. 1A). The nerves, tracts, and the neuronal cell populations that form the sources of these axons in the early mouse brain have recently been described (Easter et al., 1993; Mastick and Easter, 1996). Briefly, there are two motor nerves: the tracts are arranged orthogonally into a pair of longitudinal pathways (one dorsal, one ventral) and several dorsoventral ones, and there are eight sources. More details will be given in the appropriate section of Results. In summary, the loss of Wnt-1 function results in the early disruption of brain morphogenesis and in the subsequent deletion of large regions of the mature brain. In previous reports, the extent of the early disruptions could