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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00641

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Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain

Alexandre Pattyn1,*,{dagger}, Anna Vallstedt1,*, Jose M. Dias1, Maike Sander2,{ddagger} and Johan Ericson1,§

1 Department of Cell and Molecular Biology, Karolinska Institute, S-171 77 Stockholm, Sweden
2 Center for Molecular Neurobiology, Martinistrasse 85, 20251 Hamburg, Germany



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  		Fig. 1. Spatial generation of somatic- and visceral motoneurons in the hindbrain. (A) Schematic drawing of the embryonic hindbrain showing the distribution of visceral motoneurons (vMNs) (red) and somatic motoneurons (sMNs) (orange) along the AP axis. vMNs are generated in rhombomeres (r) 2-8 while sMNs are generated in r5 and r7-8 (Lumsden and Krumlauf, 1996Go; Cordes et al., 2001). (B-O) Transverse sections through r7 and r3 of wild-type (wt) embryos at E10.5. In r7, sMNs express Isl1/2 (B) and Hb9 (D), and are generated from the pMNs domain that express Olig2 (D,F) Nkx6.1 (J), Nkx6.2 (H), Pax6 (F) but not Nkx2.2 or Irx3 (B,L). In r3 and r7, vMNs express Isl1 and Phox2b (B,C) and are generated from the pMNv domain that expresses Nkx2.2 (B,C), Nkx6.1 (J,K) and Nkx6.2 (H,I) but not Olig2 (D,E), Pax6 (F,G) or Irx3 (L,M). sMNs are not generated in r3 and no expression of Olig2 or Hb9 is detected (E). In r3, the expression of Pax6 (G) and Irx3 (M) extends ventrally to the dorsal boundary of Nkx2.2 expression. Chx10+ V2 neurons are detected immediately dorsal to pre-migratory Isl1+ vMNs in r3 (O). In r7, Chx10+ V2 neurons are detected dorsal to Is1+/Hb9+ sMNs (N). Ventral brackets in B,D,F,H,J,L,N indicates the pMNv domain and the dorsal bracket the pMNs domain. Brackets in C,E,G,I,K,M,O indicate pMNv domain and pre-migratory vMNs. (P,Q) Summary of ventral progenitor domains and neural subtypes generated in r7 (P) and r3 (Q).

 


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  		Fig. 2. Nkx6 proteins are required to suppress interneuron characteristics in vMNs. (A-N) Transverse sections at r2 (A-H) and r7 levels (I-N) in wt and Nkx6 mutants at E10.5. The number of cells that co-express Phox2b and Isl1 in r2 and r7 in Nkx6 mutants (B,J) is similar to controls (A,I). The initial dorsal projections of vMN axons (arrowhead) are similar in Nkx6.2+/tlz controls and in Nkx6 mutants at E10.5, as revealed by lacZ expression in Isl1+ neurons (C,D). sMNs, which express Isl1 but not Phox2b and are generated dorsal to vMNs in r7 (I), are extinguished in Nkx6 mutants (J). The expression of the progenitor HD proteins Dbx2 (G,H) and Dbx1 (E,F) expands ventrally into the Nkx2.2+ domain in Nkx6 mutants. The generation of Evx1+ V0 interneurons is ventrally extended in Nkx6 mutants (K,L), and the generation of these neurons occur at the expense of V1 and V2 interneurons and sMNs (IL) (data not shown) (Vallstedt et al., 2001Go). Most Isl1+ vMNs generated in Nkx6 mutants also express Evx1 at E10.5 (M,N). The expression of Evx1 in motoneurons appeared transient and no Isl1+/Phox2b+ cells detected at E11.5 expressed Evx1 (data not shown). (O-R) Transverse sections through r4 of wt (O), Nkx6.2tlz/tlz (P), Nkx6.1-/- (Q) and Nkx6 mutant (R) embryos. In Nkx6 mutants, most vMNs expressed Evx1 (R). No expression of Evx1 could be detected in vMNs of wt embryos (O) or in Nkx6.2tlz/tlz (P) and Nkx6.1-/- (Q) single mutants at this level.

 


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  		Fig. 3. Induction of the vMN determinant Phox2b by Nkx2.2. (A-D) Consecutive transverse sections through a stage 22 chick hindbrain electroporated with RCAS-Nkx2.2 construct. Forced expression of Nkx2.2 (A) induces expression of the vMN markers Phox2b (B) and Isl1/2 at ectopic dorsal positions (C). The expression of Shh was unaffected (D). No ectopic Isl1/2+ neurons induced in response to Nkx2.2 coexpressed the V0 neuron determinant Evx1 (data not shown). (E-H) Consecutive sections through stage 22 chick hindbrain electroporated with RCAS-Nkx6.1 construct. Misexpression of Nkx6.1 (E) induces ectopic expression of Isl1/2 (G) but not Phox2b (F) or Shh (H).

 


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  		Fig. 4. The migration of vMNs is impaired in Nkx6.1 and Nkx6 mutants. (A-F) Transverse sections through r2 of wild-type embryos at E10.5 and E11.5. The expression of Nkx6.1 (C,D) and Nkx6.2 (E,F) but not Nkx2.2 (A,B) persists in Isl1+ trigeminal vMNs as they migrate from the pMNv domain towards a more dorsal position of the hindbrain. (G,H) Dorsal view of flat-mounted hindbrain at E11.5. Caudally migrating fbMNs originate in r4 and migrate caudally through r5 (arrowhead) into r6 where they turn dorsally and settle in a dorsal position (Studer et al., 1996Go). Nkx6.1 is expressed in caudally migrating fbMNs (G), whereas Nkx6.2 only is detected only in pre-migratory fbMNs in r4 (H). Both Nkx6.1 (G) and Nkx6.2 (H) are expressed in trigeminal vMNs (indicated as N.V). The color reaction in G,H was underdeveloped to reveal the expression of Nkx6.1 and Nkx6.2 in post-mitotic neurons over the expression of these genes in ventral progenitor cells. (I-M) Transverse sections through r2 at E10.5 (I,K) and E12 (J,L) of Nkx6.2+/tlz controls (I,J) and in Nkx6 mutants (K,L) showing impaired dorsal migration of trigeminal MNs in Nkx6 mutants. The position of trigeminal MNs was determined by Isl1/2 expression and their axonal projections was visualized by the expression of lacZ. In Nkx6 mutants at E10.5 (K), Isl1/2+ cells are detected closer to the ventral midline compared with Nkx6.2+/tlz controls (I). At E12, most trigeminal MNs have reached their final position close to the trigeminal nerve exit point in Nkx6.2+/tlz controls (J), whereas many cells are positioned along the migratory pathway in Nkx6 mutants at this stage (L). Note that the trigeminal nerve exit point appears unaffected Nkx6 mutants (arrowhead in J,L), but that cells settle in an aberrant ventral position in Nkx6 mutants compared with controls (J,L). (M) Quantification of vMN migratory defects in r2 of Nkx6.1 and Nkx6 mutant mice. The position of migrating Isl1+ cells along their migratory route was assessed by arbitrarily dividing r2 into four equivalent zones (indicated as z1-z4 in K,L) between the site of generation and the trigeminal nerve exit point (arrowhead in J,L). The percentage of Isl1+ cells located in each zone at E10.5 and E12 in wild-type controls, Nkx6.1 single mutants and Nkx6 mutants is indicated (M). No migratory defects were observed in Nkx6.2 single mutant mice (data not shown). (N-S) Dorsal view of flat-mounted hindbrains showing Isl1 expression in wild-type (N,Q), Nkx6.1 mutants (O,R) and Nkx6 mutants (P,S) at E11.5 and E13.5. In wild-type embryos at E11.5, Isl+ fbMNs generated in r4 have initiated their caudal migration through r5 into r6 (N), where they accumulate and settle in a lateral position at E13.5 (Q, indicated as N.VII). In Nkx6.1 mutants, fbMNs fail to migrate into r6 and instead migrate to occupy positions in r4 or r5 (O,R). In Nkx6 mutants, all fbMNs fail to initiate a caudal migration, and cells instead migrate strictly dorsally within r4 (P,S). In wild-type embryos at E11.5, Isl1+ trigeminal MNs (indicated as N.V) in r2 and r3 have migrated away from the ventral midline towards their final settling position (N,Q). As indicated above in I-M, the migration of Isl1+ trigeminal MNs occur at a slow pace in Nkx6.1 mutants (arrowhead in O) and Nkx6 mutants (arrowhead in P).

 


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  		Fig. 5. Abnormal projection patterns of vMN axons in Nkx6 mutant mice. (A,B) Dorsal view of the hindbrain of Nkx6.2+/tlz controls (A) and Nkx6 mutants (B) showing lacZ expression (detected by X-gal staining) at E10.5. On the right side of each micrograph in A,B, a schematic summary of axonal projections of vMN subtypes is included; blue indicates trigeminal MNs (V) generated in r2-3, red indicates r4-5 derived vMNs projecting in the facial nerve (VII), pink: indicates r6-derived vMNs projecting into the glossopharyngeal nerve (IX) and green indicates vMNs projecting into the vagal nerve (X). Open circles indicate the axonal exit point of vMN subtypes. In Nkx6 mutants, most vMNs generated in the caudal hindbrain fail to project out of the neural tube through their normal exit points in r6 and r7. Instead, axons turn and extend either in caudal or rostral directions within the neural tube (A,B; data not shown). Axonal projections of vMNs into the VIIth and Vth nerve is less affected and most axons converge at their respective exit point. In contrast to controls, few axons in Nkx6 mutants have at this stage exited the neural tube (A,B). A significant number of r3-derived trigeminal MNs at E10.5, arbitrarily project caudally towards the exit point in r4, rather than their normal exit point in r2. (C-F) Lateral view of E13.5 embryos showing lacZ expression in vMN axons in Nkx6.2+/tlz controls (C) and Nkx6 mutants (D). In Nkx6.2+/tlz embryos, the normal pattern of peripheral projections of the trigeminal (V), facial (VII), glossopharyngeal (IX) and vagal (X) nerves is detected. In Nkx6 mutants (D), lacZ+ projections of vMNs into the trigeminal (V), and facial (VII) nerves are severely impaired, although the overall shape of the facial nerve resembled that of control embryos. At more caudal levels of Nkx6 mutants, no lacZ+ axonal projections into the vagal (X) and glossopharyngeal nerves (IX) are detected. Schematic summary of axonal projections in control (Nkx6.2+/tlz) and Nkx6 mutants at E13.5 (E,F). (G,H) Transverse sections through the brainstem of wild type (G,I,K,M) and Nkx6 mutants (H,J,L,N) at E16.5, hybridized with a peripherin probe to visualize MN nuclei. Sections are shown in an anterior (upwards) to posterior (downwards) direction. In Nkx6 mutants, the trigeminal (N.V; H) and facial (N.VII; J) nuclei are reduced in size compared with controls (G,K) and the facial nucleus is displaced rostrally (J, compare with wild type in K). The nuclei of the vagal nerve, nucleus ambiguus (N.A) and the dorsal motor nucleus (dmnX), are absent in Nkx6 mutants (N, compare with wild type in M). In addition, sMNs of the abducens (N.VI; I) and the hypoglossal (N.XII; M) nuclei are missing in Nkx6 mutants (J,N).

 


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  		Fig. 6. Olig2 is expressed but sMNs are missing in the hindbrain of Nkx6 mutants. (A-R) Transverse sections through r7 or r4 of wild type and Nkx6 mutant embryos at E10 (A-H), E11.5 (I-J) or E10.75 (K-R). (A-H) In r7, the expression of Olig2 is similar in controls and Nkx6 mutants at E10 (A,B). Hb9+ sMNs are detected in controls (A) but not Nkx6 mutants (B). The pattern of Nkx2.2, Pax6 and Irx3 is similar in controls and Nkx6 mutants in r7 at E10 (C-F). The expression of Dbx2 is expanded ventrally and ectopically expressed in the pMNs domain (G,H). By E11.5, the expression of Olig2 is extinguished at the level of r7 in Nkx6 mutants (I,J). (K-R) In r4 of wild-type embryos at E10.75, sMNs are not generated and no expression of Olig2 or Hb9 can be detected (K). In Nkx6 mutants, Olig2 is expressed ectopically in r4, but no Hb9+ neurons are detected (L). Most ectopic Olig2+ cells in r4 of Nkx6 mutants co-express Pax6 and Nkx2.2 at E10.75 (M,N). No expression of the oligodendrocyte precursor cell markers Sox10 or Pdgfra is detected either in controls (O,Q) or in Nkx6 mutants (P,R) at E10.75.

 


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  		Fig. 7. Model for the role of Nkx proteins in somatic- and visceral MN generation. (A) sMN generation: The combinatorial expression of Nkx6 proteins (Nkx6.1 and Nkx6.2), Olig2 and Pax6 act to suppress cells in the pMNs domain from undertaking other ventral differentiation programs; Nkx6.1 and Nkx6.2 suppress V0 and V1 fate (Briscoe et al., 2000Go; Vallstedt et al., 2001Go), Olig2 suppresses V2 fate (Novitch et al., 2001Go; Zhou and Anderson, 2002Go) and Pax6 blocks vMN fate (Ericson et al., 1997Go). In addition to its role in suppressing V2 fate, Olig2 also promote cells to progress along the sMN differentiation pathway (Novitch et al., 2001Go; Zhou and Andersson). In part, Nkx6.1 and Nkx6.2 promote sMN generation by acting upstream of Olig2 (Novitch et al., 2001Go) (this study), but Nkx6 proteins also act in parallel with Olig2 in the progression of sMN fate determination. (B) vMN generation: The combinatorial activity of Nkx6 and Nkx2 class proteins in the pMNv domain suppress more dorsal sMN and interneuron differentiation programs. Nkx6.1 and Nkx6.2 suppress V0 and V1 fate (Vallstedt et al., 2001Go) (this study), while Nkx2.2 and Nkx2.9 suppress sMN and V2 neuronal fate (Briscoe et al., 1999Go; Briscoe et al., 2000Go; Pabst et al., 2003Go). In the pMNv domain, Nkx6 proteins are dispensable for the progression of vMN differentiation, and Nkx2 class proteins mediate the activation of the vMN determinant Phox2b (Pattyn et al., 2000Go). Once induced, Phox2b and Nkx2 proteins may also cooperate in subsequent steps of vMN fate determination (Dubreuil et al., 2002Go). For further details, see text.

 

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© The Company of Biologists Ltd 2003