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First published online 15 December 2008
doi: 10.1242/dev.027193

Development 136, 295-305 (2009)
Published by The Company of Biologists 2009
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The bHLH transcription factor Olig3 marks the dorsal neuroepithelium of the hindbrain and is essential for the development of brainstem nuclei

Robert Storm1,*, Justyna Cholewa-Waclaw1,*, Katja Reuter1,*, Dominique Bröhl1, Martin Sieber1,{dagger}, Mathias Treier2, Thomas Müller1 and Carmen Birchmeier1,{ddagger}

1 Max-Delbrück-Centrum for Molecular Medicine, Robert-Rössle-Strasse 10, 13125 Berlin, Germany.
2 EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany.


Figure 1
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  		Fig. 1. Characterization of neuronal subtypes that arise from Olig3+ cells in the mouse dorsal hindbrain. (A-E) Immunohistological analysis of Olig3+ cells on consecutive sections of the dorsal alar plate of rhombomere 7 at E11.5. Olig3+ cells were observed in a broad domain of the ventricular zone. See Fig. S1A in the supplementary material for an explanation of the sector displayed. Immunohistological analysis of (A) Olig3, Ki67 and Lbx1, (B) Olig3 and Math1, (C) Olig3 and Ngn1, (D) Olig3, Mash1 and Ptf1a, (E) Olig3 and Ptf1a. Note that D and E show the same section; in E, the Mash1 signal was removed, and the Ptf1a signal is shown in red. (F-H,J-L) Analysis of neuronal types generated by Olig3+ cells using genetic lineage tracing in rhombomere 7 (F-H) and rhombomeres 4-6 (J-L). Recombination was induced in Olig3CreERT2/+; Rosa26R mice at E9.5 by tamoxifen; cells that expressed the active lacZ gene were identified using anti-β-Gal antibodies. Neuronal types were defined using antibodies against Lhx2/9 (F,J), Lhx1/5 and Foxd3 (G), Tlx3 (H,K), and Lhx1/5 and Lbx1 (L). Note that J and K show the same section, which was stained for β-Gal, Lhx2/9 and Tlx3; J and K display β-Gal/Lhx2/9 and β-Gal/Tlx3 signals, respectively. (I,M) The distinct dorsal ventricular zones and neuronal subtypes of rhombomeres 7 (I) and 4-6 (M) (Sieber et al., 2007Go). Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Olig3 is required to determine the fate of class A neurons in rhombomere 7. Immunohistological (A-F,I-P) and in situ hybridization (G,H) analyses of the alar plate of rhombomere 7 of control and Olig3 mutant mice at E11.5. (A,B) In control animals (A), Lbx1+ neurons were restricted to the ventral alar plate, and Lhx2/9+ neurons (dA1) arose at the dorsal lip. In Olig3 mutant mice (B), Lbx1+ neurons arose throughout the entire alar plate, and Lhx2/9+ neurons (dA1*) co-expressed Lbx1. (C-F) Pax2 and Lhx1/5 (C,D) and Lbx1 and Pax2 (E,F) expression. (G,H) Analysis using a Gad1-specific probe. (I-P) Analyses using antibodies against Math1 (I,J), Ngn1 and Lhx1/5 (K,L), Ptf1a and Lhx1/5 (M,N) and Pax7 and Lbx1 (O,P). Note the expanded expression of Ptf1a in the Olig3 mutant mice. Distinct neuronal subtypes are indicated. Scale bars: 50 µm.

 

Figure 3
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  		Fig. 3. Olig3 is required to determine the fate of class A neurons in rhombomere 4. Immunohistological analyses (antibodies as labeled) of the alar plate of rhombomere 4 of control (Olig3+/-) and Olig3 homozygous mutant mice at E11.5. (A,B) In control animals, Lbx1+ neurons were restricted to the ventral alar plate. In Olig3 mutant mice, ectopic Lbx1+ neurons were present. (C,D) In Olig3 mutant mice, Tlx3+ (dA3*) neurons were generated in reduced numbers and co-expressed Lbx1. (E,F) Phox2b+ dA3 neurons were not present in Olig3 mutant mice, and the expression domain of Ptf1a was expanded. (G-J) In the dorsal alar plate of Olig3 mutant mice, Lhx1/5+/Lbx1+, Lhx1/5+/Lbx1- and Lbx1+/Pax2+ neurons arose in an apparently intermingled manner. Note that G,I and H,J show identical sections, which were stained with antibodies against Lbx1, Pax2 and Lhx1/5; G,H and I,J display Lbx1/Pax2 and Lbx1/Lhx1/5 signals, respectively. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. Genetic lineage tracing in heterozygous and homozygous Olig3 mutant mice. (A) Strategy used to generate the Olig3CreERT2 allele. Schematic representation of the wild-type Olig3 locus, the targeting vector, and the mutant Olig3 alleles before (Olig3CreERT2neo) and after (Olig3CreERT2) removal of the neomycin (neo) cassette. The coding exon of Olig3 (red) was interrupted by the insertion of a CreERT2-FRT-neo-FRT cassette. Indicated are CreERT2 (yellow) and the neo resistance cassette surrounded by FRT sequences (FRT-neo-FRT); in addition, a diphtheria toxin A (DTA, light green) cassette was included for negative selection. (B) Immunohistological analysis of rhombomere 7 in Olig3CreERT2/+ mice at E11.5 using antibodies against Cre and Olig3. (C-J) Analysis of the medulla oblongata and pons of control (Olig3CreERT2/+; Rosa26R) and Olig3 mutant (Olig3CreERT2/-; Rosa26R) mice at E18.5. Recombination was induced at E10.5 by tamoxifen, and expression of the active lacZ gene was identified by X-Gal staining (blue). (K,L) Immunohistological analysis using antibodies against Pax2 and NF68 (Nefl). To improve the visibility of neurons, a false color was assigned to the black background of the original photograph. Arrowheads and arrows indicate the solitary (sol) and spinal trigeminal (spV) tracts, respectively. Cu, cuneate nucleus; ECu, external cuneate nucleus; ION, inferior olivary nucleus; NTS, nucleus of the solitary tract; LRt, lateral reticular nucleus; PB, parabrachial nucleus; PGN, pontine gray nucleus; RTN, reticulotegmental nucleus; CB, cerebellum; EGL, external granular layer. Scale bars: 50 µm in B; 200 µm in D,J,L.

 

Figure 5
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  		Fig. 5. dA4 neurons and their derivative, the inferior olivary nucleus, are absent in Olig3 mutant mice. (A,B,D,E,F,H) Immunohistological analysis of control (A,B,D) and Olig3 mutant (E,F,H) mice using antibodies against Brn3a and Foxd3. In control mice at E11.5 (A), Brn3a+/Foxd3+ dA4 neurons emerged in the dorsal alar plate. Foxd3/Brn3a co-expression marks specifically dA4; other neuronal types expressed either Foxd3 (a ventral population, asterisk) or Brn3a (dorsal neuronal subtypes). At E12.5 (B), a stream of Foxd3+/Brn3a+ dA4 neurons (arrowheads) extended ventrally and appeared to assemble close the ventral midline (arrow). In Olig3 mutant mice at E11.5 (E) and at E12.5 (F), Foxd3+/Brn3a+ dA4 neurons were absent. At E18.5. Foxd3+/Brn3a+ neurons were located in the inferior olivary nucleus of control mice (arrow, D), but were absent in Olig3 mutants (H). (C,G) In situ hybridization analysis of control (C) and Olig3 mutant (G) mice at E15.5 using a Foxd3-specific probe (arrow in C). The insets show the whole-mount in situ hybridization of the corresponding hindbrains prior to sectioning. Scale bars: 50 µm in F,G; 100 µm in H.

 

Figure 6
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  		Fig. 6. Cooperation of Olig3 and Ptf1a and permissive functions of Olig3 in the determination of the Foxd3+ dA4 fate. (A-C,E-G) Chick hindbrains electroporated with mouse Olig3 (A-C,F), Ptf1a (E), Olig3 and Ptf1a (G). Effects were assessed by immunohistochemistry using antibodies against Lbx1 (A) or Pax2 (B), or by in situ hybridization using probes specific for Ptf1a (C) or Foxd3 (E-G). In addition, a GFP vector was co-electroporated to identify electroporated cells. Note that A and B show the same section stained with antibodies against Lbx1, Pax2 and GFP; A and B display the Lbx1/GFP and Pax2/GFP signals, respectively. (D,H) Quantification of cells that expressed particular transcription factors in the dorsal part of the chick caudal medulla oblongata. (I-O) Comparison of the dorsal alar plate in control (I,M), Olig3 mutant (J,N), Olig3; Lbx1 double-mutant (K,O) and Lbx1 mutant (L) mice, using antibodies against Foxd3 and Pax2 (I-L) and Foxd3 and Phox2b (M-O). The dashed line indicates the ventral limit of Oligo3 expression. (P) Summary of neuronal types observed in control, Olig3 and Olig3; Lbx1 mutant mice. The colors indicate the neural subtypes defined in Fig. 1I by their transcription factor code. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. dA3 neurons and their derivative, the nucleus of the solitary tract, are not generated in Olig3 mutant mice. The alar plate of rhombomere 7 of control (A-C) and Olig3 mutant (D-F) mice at E10.5 (A,D), E13.5 (B,E) and E18.5 (C,F). Arrows indicate vagal motoneurons. (A,D) Immunohistological analysis using antibodies against Phox2b and Tlx3. In control embryos at E10.5, Phox2b+/Tlx3+ marked dA3 neurons (bracket), Phox2b+/Tlx3- marked vagal motoneurons (arrow), and Phox2b-/Tlx3+ marked dB3 neurons (bracket). In Olig3 mutant mice, dA3 neurons were absent. (B,E) In situ hybridization using a Phox2b-specific probe. In control embryos at E13.5, dA3 neurons (asterisk) were positioned dorsal to the vagal motoneurons (arrow). dA3 neurons were absent in Olig3 mutant mice. (C,F) Immunohistological analysis using antibodies against Tlx3, Phox2b and NF68. In Olig3 mutant mice, the Phox2b+/Tlx3+ neurons (inset in C) of the nucleus of the solitary tract were absent, but vagal motoneurons (arrows) were present. Scale bars: 100 µm in D,E; 200 µm in F.

 

Figure 8
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  		Fig. 8. Development of dA1 neurons in Olig3 mutant mice. Analyses of rhombomere 7 of control (A,D,G) and Olig3 mutant (B,E,H) mice. (A,B) Immunohistological analysis using anti-Lhx2/9 antibodies at E11.5. (C) Quantification of Lhx2/9+ neuron numbers. (D,E,G,H) In situ hybridization analysis using a probe specific for Barhl1. Arrows indicate external cuneate nuclei in D,E, and the lateral reticular nuclei in G,H. (F,I) Morphometric analysis of the volume of external cuneate (F) and lateral reticular nuclei (I) of control (+/-) and Olig3 mutant mice (-/-). Scale bars: 50 µm in B; 100 µm in H.

 

Figure 9
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  		Fig. 9. Summary of the changes in neuronal fate in Olig3 mutant mice. Summary of the neuronal types generated in the alar plate of control and Olig3 mutant mice in (A) rhombomeres 4-6 and (B) rhombomere 7. In Olig3 mutant mice, the fate of class A neurons was not correctly determined, and ectopic Lbx1+ neurons appear instead. (C) Model of Olig3 function in fate determination of dA4 climbing fiber neurons. Olig3 and Ptf1a cooperate to induce the dA4 fate; Olig3 exerts its function primarily by suppressing Lbx1. Ptf1a is known to suppress Tlx3 (Glasgow et al., 2005Go; Mizuguchi et al., 2006Go; Hori et al., 2008Go), and the dA4 fate might require the suppression of Lbx1 and Tlx3 by Olig3 and Ptf1a, respectively. Olig3 also appears to suppress Ptf1a; it should be noted that in normal development, Olig3 and Ptf1a are only transiently co-expressed in cells that locate to the border of the ventricular zone (VZ) and mantle zone (MZ). The colors indicate the neuronal subtypes defined in Fig. 1I by their transcription factor code.

 

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