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Initiation of facial motoneurone migration is dependent on rhombomeres 5 and 6

MRC Centre for Developmental Neurobiology, King’s College London, Guy’s Campus, London SE1 1UL, UK

View larger version (55K): [in a new window]   Fig. 1. Mouse and chick facial branchiomotor neurones undergo different migratory pathways during development. (A,C,E,G) Ventral views of flat-mounted HH stage 23 to 24 chick hindbrains and (B,D,F,H) E11.0 to E11.5 mouse hindbrains. All the panels show the region of rhombomeres (r) 4 and 5 with the floor plate (fp) to the right (basal) and the r4 exit points to the left (alar). (A,B) Retrograde rhodamine-dextran labelling of facial branchiomotor (FBM) and visceromotor neurones (VMN) in chick (A) and mouse (B). (C-H) Expression patterns of neuronal markers in chick and mouse shown by in situ hybridisation. (C,D) Chick and mouse Hoxb1 label r4 progenitors along the dorsoventral axis. In mouse, an additional mouse Hoxb1-positive domain is detected in ventral r5 (arrow in D), whereas no equivalent expression of chick Hoxb1 is found in chick r5. The inset in D shows a transverse section of mouse Hoxb1 expression at the level of r4/r5. Note expression in the mantle layer lateral to the floor plate corresponding to migrating FBM neurones. (E) Chick Isl1 is expressed in chick in ventral r4 and r5, and in migrating FBM neurones within r4. (F) In mouse, a large stream of mouse Isl1-positive cells is present in ventral r4, r5 and rostral r6 (arrow in F). (G) Chick Phox2b is expressed at high levels in FBM and VMN neurones migrating laterally within r4 and r5, respectively. (H) In mouse, mouse Phox2b is expressed in ventral r4 and in the caudally migrating FBM population (arrow in H). G and H have an additional lateral Phox2b expression, which corresponds to the intermediate neural column expanding from r2 to r6.

View larger version (28K): [in a new window]   Fig. 2. Mouse and chick hindbrain cells mix freely in short term aggregation cultures. (A,B) Confocal photomicrographs of aggregates consisting of E9.5 mouse cells from even- and odd-numbered rhombomeres (r) (even, red; odd, green) and (D,E) from E9.5 mouse and HH stage14 chick cells from the same rhombomere (mouse, red; chick, green). The bar chart in C shows a mean number of mixed and segregated aggregates. The relative proportion of each of these categories is expressed as percentage of the total number examined. Note that even-odd pairs of rhombomeres segregate from each other (r2/3, 68%; r4/5,71%), whereas cells from even pairs of rhombomeres mix freely (78%). In the bar chart in F, cells from the same rhombomere (r5; r6) in chick and mouse mix freely (81%; 83%). m/m, aggregates between mouse rhombomeres; m/c, aggregates between mouse and chick rhombomeres.

View larger version (45K): [in a new window]   Fig. 3. Mouse rhombomeres are well integrated in the chick hindbrain. (A) Schematic of a homotopic r5-r6 grafting from an E8.5 Rosa-26 lacZ transgenic mouse donor into a HH stage10 chick host. (B,D) Dorsal views of flat-mounted chimaeras after X-Gal staining at the stages indicated. In (B) a few LacZ-positive cells spread into r4 (arrow) and the posterior boundary of the mouse graft is prominent (arrowhead). In (D) the chick r4/mouse r5 boundary is totally regenerated (see arrow) and X-Gal staining is exclusively restricted to the mouse graft. (C) Dorsal view of a flat-mounted chimaeric hindbrain hybridised with mouse kreisler. mr5/6, mouse graft consisting of r5 and r6; fp, floor plate.

View larger version (54K): [in a new window]   Fig. 4. Chick motoneurones migrate into mouse r5/6 in mouse-chick chimaeras. (A,E,G) Homotopic mouse-chick grafts. (B-D,F,H) Dorsal views of flat-mounted hindbrain after in situ hybridisation with (B,C) chick Isl1 riboprobe (in blue) and B2 mouse-specific probe (B, in red), and (D,F,H) chick Hoxb1 (in blue). (B) At HH stage 20, a compact group of chick Isl1-positive cells (see arrow) has entered the anterior border of mouse r5, whereas at HH stage 24 (C) a prominent stream of chick Isl1-positive cells has invaded the mouse tissue (surrounded by red dots). The asterisk indicates that a subpopulation of r4 neurones migrates laterally. (D) A r4-specific population positive for chick Hoxb1 expression runs along the mouse floor plate included in the graft. (F) The sole presence of mouse r5 induces migration of chick Hoxb1-positive cells into the mouse graft. The inset in F shows that thick borders surround the grafted mouse r5 and includes chick Hoxb1-positive cells. (H) The right side shows a ventral protrusion of chick Hoxb1 expression through chick r5 towards the mouse tissue (labelled with a red fluorescent cell tracker). Red dots indicate the external margin of the mouse grafts.

View larger version (87K): [in a new window]   Fig. 5. Attraction of chick r4 ventral motoneurones is specific to mouse r5/6. Diagrams of r5/6 chick ablation (A), orthotopic r5/6 graft (C) and heterotopic mouse-chick graft (E). (B,D,F) Dorsal views of flat-mounted HH stage 24 hindbrains after chick Hoxb1 in situ hybridisation. (B) The most severe case in which very little regeneration of the surrounding tissue has occurred and chick Hoxb1 expression is dramatically enlarged. (D) No obvious changes in chick Hoxb1 expression resulted after cr5/6 orthotopic grafts. (F) By replacing cr5/6 with mr2/3, chimaeric embryos showed a slightly enlarged chick Hoxb1 domain, but no extension of ventral expression.

View larger version (32K): [in a new window]   Fig. 6. Chick FBM neurones reproduce a caudal and lateral migratory pathway characteristic of mouse FBM cells in mouse-chick chimaeras. (A) Schematic of ventral aspect of facial nerve and their projections into the periphery (adapted from Jacob and Guthrie, 2000). The red and green lines indicate the branchiomotor (FBM) axonal projections towards the hyoid nerve and the visceromotor (VMN) axonal projections towards the palatine nerve, respectively. (B-D) Ventral views of flat-mounted HH stage 23-24 hindbrains after retrograde labelling of facial motoneurones. Labelling of FBM and VMN subpopulations after rhodamine-dextran fills of the facial nerve (B) and of the hyoid nerve (C). An arrowhead in C marks the presence of a few neurones in r5, as previously reported (Jacob and Guthrie, 2000). Labelling of FBM neurones after rhodamine-dextran fills of the hyoid nerve in a chimaeric embryo after chick r5/6 were replaced with mouse r5/6 (D). Only a small subpopulation of FBM neurones in r4 migrates laterally (arrowheads in D), whereas the majority of FBM neurones remain either close to the ventral midline or migrate caudally. The arrow in D shows how within the caudal migration cells are oriented caudolaterally. Ba1, first branchial arch; Ba2, second branchial arch; ct, chorda tympani; ep, r4 exit point; fp, floor plate; h, hyoid nerve; Mx, maxilla; P, palatine nerve.

View larger version (98K): [in a new window]   Fig. 7. Mouse cells are unable to migrate into chick host tissue. (A,C) Homotopic E8.75 mouse to HH stage10 chick grafts including r3/4 (A) and basal r4 (C). (B,D) Dorsal views of flat-mounted hindbrains from HH stage 24 chimaeric embryos after anti-mouse Hoxb1 immunocytochemistry. The position of the grafts is encircled with a broken red line. In B, only the grafted r4 region is Hoxb1-positive. In D, the basal plate of r4 was grafted; however, chimaeric embryos show a spread of mouse cells from medial to lateral. Note the total absence of Hoxb1-positive cells in the chick surrounding tissue in B,D. The inset in D shows an example of a mouse hindbrain explant immunostained with anti-Hoxb1 antibodies. The arrow indicates Hoxb1-positive cells in r5 along the floor plate corresponding to migrating mouse FBM neurones. br4, basal r4; fp, floor plate; mr3/4, mouse rhombomeres 3 and 4; mr4, mouse rhombomere 4.