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First published online 3 September 2003
doi: 10.1242/dev.00730

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Hox3 genes coordinate mechanisms of genetic suppression and activation in the generation of branchial and somatic motoneurons

Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA

View larger version (62K): [in a new window]   Fig. 1. Expression of Hox and non-Hox genes among motoneuron subtypes in the central hindbrain. (A) Lateral view of an E11 embryo stained with TuJ1 showing the region of the central hindbrain labeled by 4, 5 and 6 to indicate the individual rhombomeres examined in this study. (B) Flat-mount preparation of an E11.5 embryo containing a targeted Hoxb1GFP reporter (white). Hoxb1GFP labels the progenitors and differentiating branchiomotoneurons (BMN) in r4 and their migratory course (arrow) towards the ventrolateral region of r6. Two classes of motoneurons, facial BMNs (green) and abducens somatic motoneurons (SMNs, red), are represented by spheres superimposed over the expression of Hoxb1GFP to illustrate their spatial relationship. (C-E) Transverse sections of E11.5 embryo spanning r4, r5 and r6 labeled with Hoxb1GFP (green), Phox2b (blue) and Isl1/2 (red). (F-H) E11.5 hindbrain flat-mount RNA in situ hybridization for Hoxa3, Hoxb3 and Hoxd3. V, Ventral; D, Dorsal.

View larger version (74K): [in a new window]   Fig. 2. Loss of Hoxa3 and Hoxd3 results in ectopic expression of Hoxb1 in r6 with normal rhombomere periodicity. (A-D) Coronal hindbrain sections, immunolabeled for Hoxb1 of E11 control and mutant embryos with incremental loss of Hoxa3 and Hoxd3 alleles. (E,F) Coronal hindbrain sections of E11 control and Hoxa3-/-d3-/- double mutant embryos labeled for Hoxb1 (red), Hoxb4 (green) and TuJ1 (blue). Hoxb4 labels the boundary between r6 and r7. The VIIth and VIIIth cranial sensory ganglia (VII/VIII) are located ventrolateral to r4 and anterior to the otic vesicle (OV), which borders r5 and r6. (G-L) Coronal hindbrain sections of E9.25 control and Hoxa3-/-d3-/- double mutant embryos labeled for Hoxb1 (G,H), Krox20 (I,J) and Hoxb4 (K,L). Hoxb1 is expressed in r4 and in migrating neural crest cells (NCC). The otic vesicle (OV) borders r5 and r6. The bracket in H highlights the ectopic expression of Hoxb1 in r6. The arrows in K and L represent the boundary between r6 and r7.

View larger version (74K): [in a new window]   Fig. 3. Ectopic expression of Hoxb1 in r6 is associated with activation of r4-like facial branchiomotoneuron differentiation and migration. (A-F) Transverse sections of ventral r6 in E11.5 control (A,C,E) and Hoxa3-/-d3-/- double mutant embryos (B,D,F) labeled for Hoxb1 (green), Nkx2.2 (red), Isl1/2 (red) and Phox2b (green). The arrows in B, D, F represent ectopic progenitors in the ventricular layer of the neuroepithelium in r6. (G,H) Transverse sections of ventral r6 and r7 in E11.5 control (G,I) and Hoxa3-/-d3-/- double mutant embryos (H,J) labeled for Hoxb1 (green) and Hoxb4 (red). (K,L) Coronal sections of E10.5 control and Hoxa3-/-d3-/- double mutant embryos labeled for Hoxb1 (green) and Hoxb4 (red). The arrows in J and L represent ectopic Hoxb1-expressing cells in r7 that have presumably migrated from r6.

View larger version (109K): [in a new window]   Fig. 4. Hox3 and Pax6 are necessary for the development of somatic motoneurons in r5. (A-C) Transverse sections of ventral r5 in E11.25-E11.5 control, Hoxa3-/-b3-/- double and Pax6Sey/Sey mutant embryos labeled for HB9 (green) and Phox2b (red). The SMNs are missing in both Hoxa3-/-b3-/- double and Pax6Sey/Sey mutant embryos. The ventrolateral expression of Phox2b in both mutant embryos is spared, suggesting that the early differentiation of visceromotoneurons in r5 is unaffected by either mutations. (D-I) Characterization of ventral progenitors and neuronal subtypes in transverse sections of ventral r5 in control E10.25 embryos prior to the migration of r4-derived BMNs. The progenitors for V3 interneurons, SMNs and V2 interneurons are characterized by the expression of Nkx2.2 (red), low and high Pax6 (green) levels, respectively (D). Olig2 (green) labels the pSMN domain (red; green fluorescent intensity of Olig2 masks the low red fluorescence of Pax6 staining), immediately ventral to the high Pax6 pV2 domain (E). pSMN domain and SMNs are labeled by HB9 (red) and Olig2 (green), respectively (F). The SMNs and the V3 interneurons can be distinguished from each other by their non-overlapping expression of HB9 (red) and Phox2b (green), respectively (G). The V2 interneurons can be distinguished from the HB9-expressing SMNs (red) by their specific expression of Chx10 (green). A CFP reporter for Hoxa3 (green) labels all cells in r5 with relatively high expression levels among postmitotic cells, which include the HB9-expressing SMNs (red, I).

View larger version (93K): [in a new window]   Fig. 5. Specification of somatic motoneurons in r5 is regulated by Hoxa3 and Hoxb3 in a gene dose-dependent manner. (A-D) Transverse sections of ventral r5 in E10.25 embryos harboring various mutant alleles in Hoxa3 and Hoxb3 labeled for Olig2 (green) and HB9 (red); (E-H) TUNEL (red), activated caspase 3 (green, CASP3) and the mitotic marker, MPM2 (blue). (I-P) Transverse sections of ventral r5 in E10.25 control and Hoxa3-/-b3-/- double mutant embryos labeled for Pax6 (green) and Nkx2.2 (red) (I,M), Phox2b (green) and Isl1/2 (red) (J,N), Chx10 (green) and Isl1/2 (red), and Phox2b (blue) (K,O); and E11.5 control and Hoxa3-/-b3-/- double mutant embryos labeled for HB9 (green) and Chx10 (red) (L,P). Cell counts for Chx10-expressing V2 interneurons were determined for representative control (Hoxa3b3-double heterozygote) and mutant (Hoxa3b3-double homozygote) embryos. Values for the control, 40.0± 1.9 (mean/side± s.e.m.), versus double-mutant, 61.6± 2.3, were statistically significant (P<0.005). The arrows in panels L and P represent clusters of HB9- and Chx10-expressing SMNs and V2 interneurons, respectively.

View larger version (85K): [in a new window]   Fig. 6. Hoxa3 and Hoxb3 are necessary for the formation of the abducens nucleus and normal target innervation. (A-D) Transverse sections of the upper medulla in E18 control and Hoxa3-/-b3-/- double mutant embryos labeled for the neuron-specific nuclear marker, NeuN (A,B) and choline acetyltransferase (ChAT) (C,D). In the control embryo, the region of the abducens nucleus (dotted outline) can be identified by its stereotypic relationship with the axons of the genu of the facial nucleus (arrow; devoid of NeuN expression) and the expression of ChAT (A,C). In the Hoxa3-/-b3-/- double mutant embryo, the loss of ChAT expression in the region normally occupied by the abducens nucleus is not associated with the loss of NeuN-expressing neurons and the formation of the genu (B,D). (E-J) Transverse section through the proximal lateral rectus muscle in E18 control and Hoxa3-/-b3-/- double mutant embryos labeled for TuJ1 (green) and -bungarotoxin binding (BTX, red). (I,J) Merged images of E,G and F,H, respectively.

View larger version (37K): [in a new window]   Fig. 7. The paralogous Hox3 genes play two crucial roles in neuronal fate specification. (A,B) Along the AP axis of the hindbrain, the paralogous Hox3 genes are required to suppress the expression of Hoxb1 (green) in r6. The loss of Hox3 genes results in the ectopic expression of Hoxb1 associated with the activation of r4-like facial BMN differentiation and migration program in r6 - characteristic of a homeotic transformation. Although the Hox3 genes do not influence r4 directly, the observation that it is required to genetically suppress the r4-program in r6 ensures that r4 maintains its unique identity. The Hox3 genes thus influence the identities of at least r4, r5 and r6 during hindbrain development. (C,D) Along the DV axis of r5, the combined functions of Hoxa3 and Hoxb3 are necessary for the specification of somatic motoneuron progenitors (pSMN) of the abducens nucleus. Mutations of Hoxa3 and Hoxb3 are associated with the ectopic expression of the more dorsal high expression of the Pax6 V2 interneuron progenitor (pV2) domain (dark green) into more ventral the pSMN domain (light green). Subsequently, V2 interneurons are ectopic in the domain normally occupied by SMNs.