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First published online 1 October 2003
doi: 10.1242/dev.00802

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Neuronal defects in the hindbrain of Hoxa1, Hoxb1 and Hoxb2 mutants reflect regulatory interactions among these Hox genes

Anthony Gavalas1,*,{dagger}, Christiana Ruhrberg1,*,{ddagger}, Jean Livet2, Christopher E. Henderson2 and Robb Krumlauf1,3,§

1 Division of Developmental Neurobiology, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
2 INSERM U.382, Developmental Biology Institute of Marseille (CNRS-INSERM-Univ. Mediterranee) Marseille, France
3 Stowers Institute for Medical Research, 1000 East 50th Street, Kansas City, MO 64110, USA



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  		Fig. 1. Expression of Hoxb1 and Hoxb2 in the developing mouse hindbrain. (A-C) Ventricular views of flat-mounted wild-type mouse hindbrains stained with a Hoxb1 riboprobe at 10.5 dpc (A), 11.5 dpc (B) and 12.5 dpc (C). (D-H) Ventricular views of flat-mounted wild-type mouse hindbrains stained with a Hoxb2 riboprobe at 10.5 dpc (E), 11.5 dpc (F), 12.5 dpc (G), 13.5 dpc (H) and 14.5 dpc (D). (A-C) Between 10.5 dpc and 12.5 dpc, expression of Hoxb1 was restricted to rhombomere 4. It was upregulated in ventral and dorsal columns (black arrows). At 11.5 dpc (B), the ventral expression domain was further subdivided into two adjacent stripes. At 12.5 dpc (C), Hoxb1 expression was generally downregulated, but remained stronger in the ventral and dorsal columns. (D-H) Between 10.5 and 14.5 dpc, expression of Hoxb2 resolved into a series of longitudinal stripes that persisted longer than that of Hoxb1. At 10.5 dpc, Hoxb2 expression was stronger in the r4, r5 and r6 territories, where it was upregulated in specific ventral and dorsal columns (black arrows, E) that persisted to 11.5 dpc (black arrows, F). At 11.5 dpc (F), expression in the ventral domain resolved into two adjacent stripes similar to those of Hoxb1. At 12.5 dpc (G), Hoxb2 expression was absent in a ventral territory corresponding to the path of facial branchial motoneuron (fbm) migration (white arrows). From 13.5 dpc, expression was downregulated throughout the hindbrain (H), whereas at 14.5 dpc (D), expression was upregulated in a region close to the final position of the facial motor nucleus (black arrows).

 


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  		Fig. 2. Altered neurogenesis and neuronal differentiation patterns in the Hoxa1, Hoxb1 and Hoxb2 mutant hindbrains. Ventricular views of flat-mounted wild-type (A,E,I,J), Hoxb1 (B,F,J,N), Hoxb2 (C,G,K,O) and Hoxa1 (D,H,L,P) mutant hindbrains labelled with a Math3 (A-H), Phox2b (I-L) or GATA2 (M-P) riboprobes. (A-H) At 10.5 and 11.5 dpc, Math3 expression resolved into longitudinal stripes and was particularly strong in a ventral r4 column (Vc) (black arrowheads, A) and in a medial column (Mc) extending from r4 through to r6 (bar, A). There was also some upregulation at a more dorsal column (Dc). At 11.5 dpc, the ventral r4 column segregates into two adjacent stripes (black arrowheads, E). There is no r4-specific upregulation of the ventral column in either Hoxb1 (white arrowheads, B,F) or Hoxa1 (white arrowheads, D,H) mutants. Some upregulation was evident in Hoxb2 mutants at 10.5 (black arrowheads, C), but not at 11.5 dpc (white arrowheads, G). Expression within the dorsal column extended throughout only two rhombomeres in the Hoxb1 mutants (bar, B,F) and one rhombomere in Hoxa1 mutants (bar, D,H), but appeared normal in the Hoxb2 mutants (bar, C,G). (I-L) Phox2b expression at 10.5 dpc. In the wild-type hindbrain, expression was strongest in a ventral r4 column (black arrowheads), but it was also upregulated in a medial column extending from r2 to r6, and a dorsal column extending from r4 posteriorly (I). The medial and dorsal columns overlap in three rhombomeres: r4, r5 and r6 (black bar, I). In Hoxb1 (J) and Hoxa1 (L) mutants, there was no r4-specific upregulation (white arrowheads). The medial and dorsal columns overlap by only two rhombomeres in Hoxb1 mutants (J, bar) and one rhombomere in Hoxa1 mutants (L, bar). In Hoxb2 mutants (K), expression levels in ventral r4 were lower than in wild type, but remained elevated when compared with neighbouring rhombomeres (black arrowheads). (M-P) Gata2 expression at 10.25 dpc. In wild-type hindbrain, Gata2 expression is strongly upregulated in ventral r4 (M, black arrowheads). Gata2 expression is absent in this domain in Hoxb1 mutants (N, white arrowheads) and slightly reduced in the Hoxb2 mutants (O, black arrowheads). Both the width and the strength of this domain were greatly reduced in the Hoxa1 mutants (P, black arrowheads).

 


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  		Fig. 3. Abnormal migration of r4-derived motoneurons in Hoxa1, Hoxb1 and Hoxb2 mutant hindbrains. (A-D) Time course of Isl1 expression in wild-type flat-mounted hindbrains between 10.5 and 11.5 dpc (ventricular view). Expression was detected in the ventral domain of all rhombomeres, except rhombomere 1, and was strongest in r2 and r4, corresponding to the precursors of the trigeminal (Vm) and facial (VIIm) motoneurons, respectively. At 11.25 dpc, dorsal migration of the Vm and caudal migration of the VIIm began (C, black arrowheads) and continued until 11.5 dpc (D, black arrowheads) when Vm reached their final dorsal position. (E-H) Motoneuron distribution at 11.25 dpc in wild-type (E), Hoxa1 (F), Hoxb1 (G) and Hoxb2 (H) mutant flat-mounted hindbrains (ventricular view). The caudally migrating VIIm population was strongly reduced in Hoxa1 mutants (F, black arrowheads), absent in Hoxb1 mutants and depleted in Hoxb2 mutants (H, black arrowheads). The loss of the caudally migrating VIIm in the Hoxb1 mutants unmasks the abducens motoneurons (VIm) (G, black arrowheads). Note the presence of ectopic motor nuclei in r4 in all three mutants (F-H asterisks). (I-P) Ventricular (I-L) and pial (M-P) views of motoneuron distribution at 12.5 dpc in wild-type (I, M), Hoxa1 (J, N), Hoxb1 (K, O) and Hoxb2 (L, P) mutant flat-mounted hindbrains. At this stage, VIIm normally started migrating caudally and dorsally reaching the pial side of the hindbrain (I, M, black arrowheads). In the Hoxa1 mutants, caudal migration was sparse and no VIIm had reached the pial side at this stage (N, white arrowheads). Some migrating motoneurons turned dorsally prematurely (J, black arrowheads). In the Hoxb1 mutants, VIIm did not follow the normal migratory pathway (K,O, white arrowheads). This population was reduced in the Hoxb2 mutants. At this stage, all three mutants formed ectopic motor nuclei in r4 (J-K; asterisks in N-P). (Q-T) Ventricular views of the Isl1 expression pattern in flat-mounted Hoxb2 mutant hindbrains at 12.5 dpc. The VIIm population following the normal migratory pathway was variably reduced (black arrowheads). The ectopic nuclei were also variable (asterisks) in both their density and AP position.

 


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  		Fig. 4. Comparably low Lhx4 and Er81 expression in the facial motor nucleus of Hoxb2 mutants. Ventricular view of Lhx4 (A,B) and Er81 (C,D) expression in the hindbrain of Hoxb2 heterozygous (A,C) and mutant (B,D) embryos at 14.5 dpc. Lhx4 is normally expressed in the trigeminal (V), abducens (VI), facial (VII), nucleus ambiguus (Amb) and dorsal r4/r5-derived [(VIIb) which are likely to include the superior salivatory nucleus] motoneurons (A). Er81 is expressed only in the facial motor nucleus and the trigeminal nerve exit point. (C) Both genes define specific subpopulations within the facial nucleus (red circles in A,C). In Hoxb2 mutants, expression of both markers is diminished in the facial nucleus, but only Lhx4 is expressed ectopically in a dorsal population (compare arrows in B,D).

 


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  		Fig. 5. Ectopic nuclei retained expression of Phox2b in wild-type, Hoxb1 and Hoxb2 mutant hindbrains at 12.5 dpc. Ventricular (A-C) and pial (D-F) views of flat-mounted wild-type (A,D), Hoxb1 (B,E) and Hoxb2 (C,F) mutant hindbrains at 12.5 dpc. At this stage, Phox2b was expressed in both Vm and VIIm in the wild type and the mutants (D-F, black arrowheads). Ectopic nuclei retained expression of Phox2b in both Hoxb1 (E, asterisks) and Hoxb2 (F, asterisks) mutants. Note the lack of Phox2b expression in the migratory path of the facial motoneurons.

 


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  		Fig. 6. Persistent identity changes detected by altered expression of Met and Cad8 in wild-type and mutant hindbrains. (A,B) Ventricular views of Met expression in wild-type (A) and Hoxb1 (B) mutant flat-mounted hindbrains at 12.5 dpc. In the wild type, Met was expressed in the Vm trigeminal (A, arrowhead) and the superior salivatory nucleus (A, bracket) only. In the Hoxb1 mutants, there were ectopic nuclei expressing Met (B, asterisks). Note also the reduction of the superior salivatory nucleus (B, brackets). (C-H) Pial views of Cad8 expression in wild type (C,F), Hoxa1 (D,G) and Hoxb1 (E,H) flat-mounted hindbrains at 12.5 dpc (C-E) and 13.5 dpc (F-H). In wild-type hindbrains, Cad8 was expressed in the ventral half or r1, r2 and r3 (C,F) and in the VIIm (C,F, black arrowheads) at 12.5 and 13.5 dpc. At 12.5 dpc, there was a longitudinal stripe of Cad8 expression adjacent to the floor plate of r1 and r2, which extended into r3 at 13.5 dpc (C,F vertical bars). In Hoxa1 mutants, r3 expanded caudally in both 12.5 (D) and 13.5 (G) dpc (compare the vertical bar in D with C, and in G with F), and VIIm were barely detectable only at 13.5 dpc. In Hoxb1 mutants, Cad8 was ectopically expressed in r4 in a pattern reminiscent of r2 (E, r4*) at both 12.5 (E) and 13.5 (H) dpc, but no VIIm could be detected in either stage (E,H, white arrowheads).

 


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  		Fig. 7. Ectopic cell death correlates with the loss of ectopic nuclei by 14.5 dpc. (A,B) TUNEL staining in parasagittal sections of wild-type and Hoxb1 mutant hindbrains. There was ectopic cell death in the mutant (B, black arrowheads) caudally to the pontine flexure (pf). (C-F) Pial (ventricular where noted) views of Isl1 expression in wild-type (C), Hoxa1 (D), Hoxb1 (E) and Hoxb2 (F) mutant flat-mounted hindbrains at 13.5 dpc. By this stage, VIIm had normally completed their migration (C, black arrowheads). In Hoxa1 mutants, a small VIIm of variable size could be detected (D, black arrowheads), whereas the VIm was absent (D, white arrowheads). In Hoxb1 mutants, ectopic nuclei could still be detected (E, asterisks). The VIIm (E, white arrowheads), but not the VIm (E, black arrowheads) were absent. In Hoxb2 mutants, ectopic nuclei could also be detected (F, asterisks), whereas the VIm and a small VIIm were present (F, black arrowheads). (G-J) Pial (ventricular where noted) views of Isl1 expression in wild type (G), Hoxa1 (H), Hoxb1 (I) and Hoxb2 (J) mutant flat-mounted hindbrains at 14.5 dpc. In the wild type, Isl1 expression persisted in the Vm, VIm and VIIm (G, black arrowheads). In Hoxa1 mutants, the VIIm were either missing or rudimentary (H, white and black arrowheads), whereas the VIm was absent (H, white arrowheads). In Hoxb1 and Hoxb2 mutants, no ectopic nuclei could be detected. The VIIm was absent in Hoxb1 mutants (I, white arrowheads) and reduced in Hoxb2 mutants (J, black arrowheads), while the VIm was present in both mutants (I,J, black arrowheads).

 


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  		Fig. 8. Bax inactivation could prolong the survival of ectopic nuclei. (A-D) Ventricular views of Isl1 expression in wild-type (A), Bax (B), Hoxb1 (C) and Hoxb1/Bax (D) flat-mounted mutant hindbrains at 14.5 dpc. In both wild-type and Bax mutants, the Vm, VIm and VIIm could be detected (A,B, black arrows). In Hoxb1 mutants only the Vm and VIm could be detected (C, black arrowheads), whereas the VIIm was absent (C, white arrowheads). In Hoxb1/Bax mutants, the Vm and the VIm could be detected (D, black arrowheads), but not the VIIm (D, white arrowheads). In Bax and Hoxb1/Bax mutants there was an additional ventral column of ectopic Isl1-positive cells in the caudal hindbrain (B,D, asterisks).

 


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  		Fig. 9. Regulatory interactions among the Hoxa1, Hoxb1 and Hoxb2 genes. (A-F) Dorsal views of Hoxb1 expression in wild-type (A,C,E) and Hoxb2 mutants (B,D,F) at 8.5 (A,B), 9.5 (C,D) and 10.5 (E,F) dpc. Expression of Hoxb1 is initiated (A,B) but not properly maintained (C-F) in the Hoxb2 mutants. (G-O) Expression of a Hoxb1 lacZ transgene (HL5) in wild type (G,J,M), and Hoxb2 mutants (H,I,K,L,N,O) at 9.5 (G-I), 10.5 (J-L) and 12.5 (M-O) dpc in side views of whole mount embryos (G-I) and dorsal views of flat mounted hindbrains (J-O). Expression of the HL5 transgene is variable in the Hoxb2 mutants (H,I,K,L,N,O). Note also that lacZ expression is maintained in migrating fbms in the wild type (J,M) and in a proportion of migrating fbms in some (K,N), but not all (L,O), Hoxb2 embryos. (P) The regulatory interactions among the three genes. Hoxb1 and Hoxa1 expression is initiated through the action of retinoids (green line). In turn, Hoxa1, Hoxb1 and co-factors Pbx/Meis establish the expression of the latter in the r4 territory (orange line), which is subsequently maintained through Hoxb1 autoregulation (red arrow). Hoxb2 directly or indirectly (broken blue arrow) feedback upon Hoxb1 to maintain its expression in r4. An indirect contribution could be achieved through a requirement for Hoxb2 in the maintenance of the r4 territory. Hoxb1 and Hoxb2 synergise to regulate target genes pertaining to segmental identity and neurogenesis in r4.

 


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  		Fig. 10. Fate of hindbrain motoneuron precursors in Hoxa1, Hoxb1 and Hoxb2 mutants. A schematised view of early (10.5 dpc, top row) axial identity and subsequent (12.5, bottom row) fate of hindbrain motoneuron precursors in wild type, Hoxa1, Hoxb1 and Hoxb2 embryos. At 10.5 dpc, r2-r5 have normally acquired specific identities and the corresponding motoneuron precursors do not mix. The segregated precursor populations will give rise to the trigeminal (V, derived from r2 and r3), abducens (VI, derived from r5) and facial (VII derived from r4) motor nuclei. In Hoxa1 mutants, the r3/r4 boundary does not form and most of the r5 territory is lost, resulting in a single rhombomere often referred to as rx. Some of the presumptive r4 precursors have acquired a partial r2 identity and mix with r3 precursors. The latter form an ectopic transient motor nucleus (asterisk) and sometimes a small facial (VII) motor nucleus. As a result of r5 strong reduction, the abducens motor nucleus (VIm) is lost. In Hoxb1 mutants, presumptive r4 precursors are misspecified to an r2-like identity. As a result, they all migrate in a trigeminal like manner and form an ectopic, but transient, motor nucleus (asterisk), which is cleared by cell death. In Hoxb2 mutants, some r4 precursors have acquired an r2-like identity, and as a result a transient ectopic motor nucleus (asterisk) is formed in addition to the facial motor nucleus.

 

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© The Company of Biologists Ltd 2003