Deleted in colorectal cancer (DCC) regulates the migration of luteinizing hormone-releasing hormone neurons to the basal forebrain

Abstract

Luteinizing hormone-releasing hormone (LHRH) neurons migrate from the vomeronasal organ (VNO) to the forebrain in all mammals studied. In mice, most LHRH neuron migration is dependent on axons that originate in the VNO but bypass the olfactory bulb and project into the basal forebrain. Thus, cues that regulate the trajectories of these vomeronasal axons are candidates for determining the destination of LHRH neurons. Using in situ hybridization techniques, we examined the expression of Deleted in colorectal cancer (DCC), a vertebrate receptor for the guidance molecule netrin-1, during development of the olfactory system. DCC is expressed by cells in the olfactory epithelium (OE) and VNO, and in cells migrating from the OE and VNO from embryonic day 11 (E11) to E14. Some DCC(+) cells on vomeronasal axons in the nose also express LHRH. However, DCC expression is downregulated beginning at E12, so few if any LHRH neurons in the forebrain also express DCC. In rat, DCC is expressed on TAG-1(+) axons that guide migrating LHRH neurons. We therefore examined LHRH neuron migration in DCC(-/-) mice and found that trajectories of the caudal vomeronasal nerve and positions of LHRH neurons are abnormal. Fewer than the normal number of LHRH neurons are found in the basal forebrain, and many LHRH neurons are displaced into the cerebral cortex of DCC(-/-) mice. These results are consistent with the idea that DCC regulates the trajectories of a subset of vomeronasal axons that guide the migration of LHRH neurons. Loss of DCC function results in the migration of many LHRH neurons to inappropriate destinations.

Fig. 1.

DCC is expressed in the mouse embryonic olfactory system. At E11 (A) and E12 (B), in coronal sections in situhybridization reveals DCC expression in groups of cells in the olfactory epithelium (oe) and developing vomeronasal organ (vno). There are also cells that have migrated away from the VNO (arrows) and cells in the migrating mass (mm) that have migrated from the OE. Cells in the forebrain (fb) and developing olfactory bulb also express DCC. At E15 (C), DCC is no longer expressed by cells in the OE, VNO, or by migrating cells. Medial isleft, dorsal is up. Scale bar: A,B, 50 μm; C, 100 μm.

Fig. 2.

Expression of netrin-1 and DCC in mouse nose and forebrain. In the E12 forebrain, netrin-1⁺ and DCC⁺ cells are largely expressed in a complementary pattern. Netrin-1 is heavily expressed (A, open arrowhead) in the ventricular and subventricular zones of the forebrain (fb), particularly at the rostral tip of the third ventricle (v) that will become the region of the OVLT. Netrin-1 expression rapidly decreases in more dorsal regions of the developing forebrain. In contrast, DCC (B) is expressed in postmitotic zones throughout the telencephalon (B, arrowhead) and only overlaps with netrin-1⁺ in a region that will become the diagonal band of Broca. DCC is also expressed in cells in the VNO and on vomeronasal axons extending from the VNO (B, arrows). The position of the cribriform plate is shown as a dashed line. Dorsal is up, rostral is left. Scale bar, 100 μm.

Fig. 3.

DCC is expressed on TAG-1⁺vomeronasal axons in rat. At the top left is a schematic drawing of a horizontal section through the embryonic nose and forebrain. The blue box depicts a region of the VNO and OE shown in A–C. The red box outlines a region of the forebrain shown in D–F. In double-label immunofluorescence studies of horizontal sections through the nasal compartment of the E14 rat, TAG-1 immunoreactivity is detected on the nasal vomeronasal nerve (A, arrows), which originates in the vomeronasal organ and extends between the septum and the olfactory epithelium (oe). DCC is also expressed on the vomeronasal nerve (B, arrows). A double-label image reveals that most vomeronasal nerve fibers express both molecules (C). In a confocal image of a horizontal section at E16, TAG-1 (D) and DCC (E) are both expressed by caudal vomeronasal (arrows) axons growing past the forebrain (fb). Medial is right, caudal is up. DCC is expressed by a subset of LHRH cells. In sagittal sections through the nose and forebrain of an E12 mouse, fluorescence in situhybridization reveals five distinct DCC⁺ cells along the vomeronasal nerve in the nasal compartment (G, arrowheads). Fluorescence immunocytochemistry for LHRH on the same section as in G, reveals four LHRH⁺ neurons (H, arrows). In a double-label image (I) showing both DCC and LHRH fluorescence, two cells (arrows andarrowheads) are DCC⁺/LHRH⁺, three cells are DCC⁺/LHRH⁻, and two cells are DCC-/LHRH⁺. The cribriform plate is to thetop left, and the VNO is to the bottom right. Scale bar: A–C, 50 μm;D–F, 25 μm; G–I, 10 μm.

Fig. 4.

Loss of DCC function altered LHRH neuron location in the embryonic forebrain. The percentage of LHRH neurons located in the dorsal forebrain was significantly greater in DCC homozygous mutant (−/−) embryos compared with wild-type embryos (+/+) at E13, E15, and P0. Although there was a tendency for a greater percentage of cells to be located in the dorsal forebrain of DCC^+/−, compared with DCC^+/+ mice at these ages, the difference did not reach statistical significance (A–C). The percentage of LHRH neurons located in the cortex of DCC^−/− mice compared with DCC^+/+ mice was also significantly greater at E13 and E15. There was a tendency for a greater percentage of LHRH neurons to be located in the cortex of DCC^+/− mice at E13, and this measure became significantly different from DCC^+/+ mice at E15 (D, E). *Significantly different from DCC^+/+; p < 0.05, bypost hoc Tukey–Kramer HSD.

Fig. 5.

LHRH neurons follow the caudal vomeronasal nerve. In sagittal sections through the forebrain, the trajectories of the caudal vomeronasal nerve and positions of LHRH neurons were compared in DCC^+/+, DCC^+/−, and DCC^−/− mice at E13. Immunocytochemical analysis with antibodies to peripherin show the typical turn made by the cVNN in the forebrain of wild-type mice (A, arrow) and the many defasciculated fibers growing toward the basal forebrain (arrowheads). Most LHRH neurons also turn ventrally in the forebrain of wild-type mice (B). In DCC^+/− mice (C), the peripherin⁺ cVNN also turns toward the basal forebrain, although the number of defasciculated fibers (arrowheads) and the degree of axon turning may be decreased compared with wild-type mice. The positions of LHRH neurons in heterozygous DCC mice (D) parallel the trajectories of the axons in C. In homozygous mutant DCC mice, most peripherin⁺ axons fail to turn (E, arrow), and there are few defasciculated fibers (E, arrowhead) growing to the basal forebrain. In DCC^−/− mice, LHRH neurons (F) fail to turn ventrally and migrate instead along the medial wall of the cerebral cortex. Dorsal is up, caudal is right. Scale bar, 100 μm.

Fig. 6.

Few LHRH fibers reach the median eminence (me) in the DCC^−/− basal forebrain at P0. In a sagittal section of a P0 mouse brain (A), asolid line connecting the rhinencephalic sulcus (rs) and the cortical mantle establishes a dorsal ventral boundary. The dashed line connecting the accessory olfactory bulb with the roof of the hypothalamus defines the boundary that was used to designate a more extreme subset labeled as cortex. Immunocytochemistry for LHRH in sagittal sections through the basal forebrain at P0 was performed in DCC-deficient mice and their wild-type littermates. At P0, in wild-type mice, many LHRH neurons have migrated deep into the basal forebrain, and many LHRH fibers have reached the ME (B). In contrast, in a matched section through the forebrain of a homozygous mutant littermate (C), LHRH cells are located and oriented more dorsally (compare white arrows), and only a few fibers are detectable at the ME adjacent to the pituitary (pit). The rhinencephalic sulcus that was used to help designate a dorsal boundary in conjunction with the cortical mantle in analyses at E13 and E15 (Fig. 4) continues to indicate an apparent border for LHRH neurons in DCC^−/− animals at P0.ob, Olfactory bulb; ot, optic tract;ac, anterior commissure. Dorsal is up, rostral is left. Scale bar, 200 μm.

Fig. 7.

Schematic illustration of axon trajectories and LHRH neuron positions in wild-type and DCC-deficient mice. In DCC^+/+ mice the nasal vomeronasal nerve (nVNN) originates in the VNO and splits after crossing the cribriform plate (cp). One branch grows dorsally to the accessory olfactory bulb (obVNN), and another branch grows caudally (cVNN) into the ventral forebrain. Most LHRH neurons follow the cVNN to the forebrain. In DCC^−/− mice, the nVNN branches normally into the obVNN and the cVNN. However, the majority of axons that make up the cVNN fail to make their characteristic ventral turn in DCC-deficient mice and grow into the medial wall of the cerebral cortex. Many LHRH neurons follow the aberrant trajectories of the cVNN and migrate into the cortex of DCC mutant mice.