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doi: 10.1242/10.1242/dev.00496

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Somatic motoneurone specification in the hindbrain: the influence of somite-derived signals, retinoic acid and Hoxa3

Sonia Guidato?,1, Fabrice Prin? and Sarah Guthrie?,*

? MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's College, Guy's Campus, London SE1 1UL, UK
1 Present address: Department of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK



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  		Fig. 1. Patterns of gene expression in rhombomeres and cranial motoneurones. (A) Diagram of a flat-mounted stage 28 chick embryo hindbrain and caudal half of the midbrain showing the different combinations of Lim homeobox genes expressed by each motor nucleus and the expression pattern of three Hox genes, Hoxa3, Hoxb3 and Hoxd4. Rhombomere boundaries are no longer visible at this stage but are shown for clarity. Motor nuclei: III, oculomotor; IV, trochlear; V, trigeminal; VI, abducens; VII, facial; IX, glossopharyngeal; X, vagus; XI, accessory; XII, hypoglossal [adapted, with permission, from Varela-Echavarría et al. (Varela-Echavarría et al., 1996Go)]. (B-F) Flat mounts of E6 chick midbrain and hindbrain preparations. Red lines indicate the midbrain-hindbrain boundary. Whole-mount in situ hybridisation for Islet2 (B), Hb9 (C), Hoxa3 (D), Hoxb3/Islet2 (E) and Hoxd4/Islet2 (F). mb, midbrain; hb, hindbrain; r, rhombomere; fp, floor plate; a, accessory; cva, contralateral vestibuloacoustic. Scale bar: 500 µm.

 


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  		Fig. 2. Rhombomere grafts in the pre-otic region. (A) Transplant of r3 at level of r5 (r3/r5) and of r5 at level of r3 (r5/r3). (B-N) Flat mount of E6 chick/quail chimera hindbrains in situ hybridised with probes for Islet2, Hb9, Hoxa3 or Hoxb3 as indicated. Labels in white bars above each panel indicate the type of graft and embryonic stage at which the graft was undertaken. Asterisk indicates grafted rhombomere. (G,N) Additional immunostaining of the embryos in F and M, respectively, with anti-quail cell antibodies (QCPN) to show position of the graft. (J,K) Higher magnification of H and I, respectively. (O-S) Double-immunostaining (except S; single immunostaining) on sections of an E6 embryo that had received an r5 in r3 graft at stage 10; transverse cryostat sections through r3 (O-Q) or parasagittal vibratome sections (R,S). (S) Higher magnification of R. Antibodies as indicated: QCPN, anti-quail cells; NF, anti-neurofilament; QN, anti-quail axons; Islet2, anti-Islet2. r, rhombomere; HH indicates Hamburger/Hamilton staging of embryo. White arrows indicate: (O,Q-S) axonal bundles from the graft joining the host abducens (Abd); (P) r5* Islet2-positive motoneurones. Scale bar: 580 µm for B,E,F-I,L-N; 400 µm for C,D,J,K; 240 µm for O,Q; 120 µm for P; 500 µm for R; 350 µm for S.

 


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  		Fig. 3. Rhombomere grafts in the post-otic region. (A) Transplant of r3 or r4 at level of anterior (r8a) or posterior (r8p) r8. (B-J) Flat mounts of E6 chick/quail chimera hindbrain preparations containing rhombomere grafts, in situ hybridised with probes for Islet2, Hb9, Hoxa3 or Hoxd4 as indicated by blue type. Labels in white bars above each panel indicate the type of graft and embryonic stage at which the graft was undertaken. E,G and H show only the r8 region. Asterisk indicates grafted rhombomere. Double-headed arrows indicate rostrocaudal extent of grafted rhombomere. (D) Higher magnification of C showing immunofluorescent QCPN staining (red) of grafted r3. (J) Higher magnification of I showing immunofluorescent QCPN staining (red) of grafted r4. (K-O) Double-immunostaining on transverse serial cryostat sections (level of r8p) of E6 embryo that had received an r4 in r8p graft at stage 10. Antibodies as indicated as in Fig. 2, with the addition of anti-Islet1/2 and anti-Lim3 antibodies. White arrow (K) indicates quail axons from r4* leaving the neuroepithelium ventrally and joining host hypoglossal axons. (P) Diagram of a transverse section level of r8p summarising normal Lim code in host r8p versus grafted r4*. Scale bar: 580 µm for B,C,F,I; 190 µm for E,G,H,J; 140 µm for D; 340 µm for K; 120 µm for L,N; 60 µm for M,O.

 


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  		Fig. 4. Somite or RA-loaded bead grafts. (A) Somite and RA bead grafts at the level of r2/r3, r3/r4 and r6/r7. (B-J) Flat mounts of E6 chick hindbrain preparations after somite or RA bead grafts, in situ hybridised with probes for Islet2, Hb9, Hoxa3 or Hoxd4 as indicated. Labels in white bar above each panel indicate the type of graft and embryonic stage at which the graft was undertaken. (D) Double in situ hybridisation with probes for Islet2 and Hoxd4. Asterisks indicate the grafted side and double-headed arrows indicate ectopic gene expression. (K-M,O-T) Double-immunostaining on a sagittal vibratome section (M) or transverse cryosections (K,L,O-T) of E6 chick embryos that have received a somite (M,O-T) or an RA bead graft (K,L) adjacent to the r3/r4 (O-S) or r6/r7 (T) level at stage 10. Antibodies are as indicated, abbreviations as in Figs 1, 2, 3. (M) Rhombomere boundaries are shown by a white line whereas the white asterisk highlights r4 ectopic SM axons. (O) QCPN stains the mesoderm, identifying the grafted somite (as in M). P and S are higher magnifications of O and R, respectively, showing the grafted side only. (N) Diagram of a transverse section of a chick embryo at the level of r4 summarising the normal Lim code on the control side (right) in comparison with the one on the somite/RA bead-grafted side (left). Although Islet1-positive motoneurones are present on either side, only the grafted side contains Islet2-positive/Lim3-negative motoneurones, consistent with an abducens identity for the r4 ectopic SM neurones. Note that when the somite was grafted at the level of r6/r7 the grafted side contains Islet2/Lim3-positive motoneurones consistent with a hypoglossal identity for the r7 ectopic SM neurones (see also Fig. 3P). Scale bar: 720 µm for B-D; 480 µm for E-H; 190 µm for I,J; 60 µm for L,P,S; 100 µm for M; 120 µm for K,Q,R,T.

 


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  		Fig. 5. Misexpression of Hoxa3 in the rostral hindbrain. (A) Flat mount of mouse hindbrain (E10.5) in situ hybridised for mouse Hoxa3. (B,C) Dorsal view of a chick head electroporated on the left side with tauGFP/Hoxa3 at stage 10 and incubated until E4, showing co-localisation of GFP fluorescence (B) and in situ hybridisation for Hoxa3 (C). (D-I) Flat mounts of E4 chick hindbrains electroporated at stage 12 (D-G) or 17 (H,I) with Hoxa3/tauGFP, and in situ hybridised for Hb9. Double-headed arrow indicates Hb9 induction. E and F are higher magnifications of D and G, respectively. (I) Same embryo as H following immunostaining for GFP protein to show the area of electroporation. Note that at stage 17 no Hb9 induction was observed following electroporation of tauGFP/Hoxa3. (J,K) Flat mounts of the same E6 chick hindbrain electroporated with human HOXB3/tauGFP at stage 11, in situ hybridised for Hb9 (J) and immunostained for GFP (K). Note that no induction of Hb9 was observed. Scale bar: 580 µm for A-C; 420 µm for D,G; 250 µm for E,F; 300 µm for H,I; 500 µm for J,K.

 


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  		Fig. 6. Dorsoventral patterning following Hoxa3 misexpression. (A-T) Immunostaining on transverse cryostat sections of embryos electroporated with mouse Hoxa3/tauGFP at stage 10. (A-D,E-H) Sections of two different embryos grown up to stage 27 prior to fixing. (I-P) Sections through another embryo that was fixed at stage 18. (Q-T) Sections through a second embryo fixed at stage 18. D,F,H,J-K,M-N are higher magnifications of C,E,G,I,L, respectively, showing only the electroporated side. Antibodies are as indicated. (A,E-F,J,M,Q,S) GFP natural fluorescence. K,N,R,T are the same sections as J,M,Q,S, respectively, but without the GFP signal. White asterisks (A,B,C,G,I,L,O) indicate the electroporated side. White arrows (D) indicate Islet2/Lim3-positive motoneurones. Asterisks (O,T) represent regions of repression of Pax6 and of induction of Olig2 expression, respectively. (P) r8, (S,T) r1; all other axial levels shown are r3/4. Scale bar: 120 µm for A,C,Q,T; 60 µm for D,E,G,I,L,O-P; 30 µm for F,H,J-K,M-N.

 




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