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. 2008 Jul 30:1222:42-50.
doi: 10.1016/j.brainres.2008.04.072. Epub 2008 May 6.

Expression and function of Nkx6.3 in vertebrate hindbrain

Brian P Hafler  1 , Michael Y Choi, Ramesh A Shivdasani, David H Rowitch
Affiliations

Expression and function of Nkx6.3 in vertebrate hindbrain

Brian P Hafler et al. Brain Res. .

Abstract

Homeodomain transcription factors serve important functions in organogenesis and tissue differentiation, particularly with respect to the positional identity of individual cells. The Nkx6 subfamily controls tissue differentiation in the developing central nervous system where they function as transcriptional repressor proteins. Recent work indicates that Nkx6.3 is expressed in hindbrain V2 interneurons that co-express Nkx6.1, suggesting the possibility of functional redundancy. Here, we report that Nkx6.3 expression is specific to Chx10+ V2a interneurons but not to Gata3+ V2b interneurons of the hindbrain, and that Nkx6.3 expression appears to mark cells of the prospective medullary reticular formation. Molecular analysis of Nkx6.3 null embryonic mouse hindbrain did not reveal detectable defects in progenitor markers, motor neuron or V2 interneuron sub-types. Forced expression of Nkx6.3 and Nkx6.1 promote V2 interneuron differentiation in the developing chick hindbrain. These findings indicate Nkx6.3 function is dispensable for CNS development and lead to the proposal that absence of overt defects is due to functional compensation from a related homeodomain transcription factor.

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Figures

Figure 1
Figure 1. Expression of Nkx6.3 in V2a interneurons of the embryonic CNS
(A–D) E10.5 embryo. (A) Whole mount in situ hybridization confirms Nkx6.3 expression between rhombomere 5 and rhombomere 8 (r5–8). A–P level of analysis (transverse black line at r7), telencephalon (tel), diencephalon (di), mesencephalon (mes), metencephalon (met), and myelencephalon (myel) are indicated. (B) Cartoon of transverse section at level of analysis showing prospective V2a/b and MN populations. (C) Combined double-label ISH for Nkx6.3 mRNA (pseudocolored red, cytoplasmic) and immunohistochemistry (IHC) for Lhx3 proteins (green, nuclear); (D) confocal photomicrograph showing overlap of Nkx6.3 and Lhx3 in some ventrolateral cells. (E–L) E12.5 embryo. (E) Nkx6.3 ISH in a transverse section from E12.5 caudal hindbrain. (F) Cartoon showing relative location of Nkx6.3 expression with V2a/b interneuron and motor neuron populations in the E12.5 caudal hindbrain at the level of analysis. (G–L) Double-label ISH for Nkx6.3 (red pseudocolor) and IHC with against Chx10, Gata3 and Lhx3 (green). (H, J, L) High-resolution confocal images from areas indicated within (G, I. K). (G. H) Note that (G, H) the V2a marker Chx10 and (K, L) Lhx3 are co-expressed in most Nkx6.3+ cells, whereas the V2b marker Gata3 is not co-expressed. In all panels except (A) dorsal is top, ventral is bottom, medial (m) is right and lateral (l) is to left. Scale bars: C, G, I, K 10 µM, F 100 µM, D, H, J, L 25 µM. Original magnification A x2.5, C, G, I, K x40, F x10, D, H, J, L x80.
Figure 2
Figure 2. Further characterization of Nkx6.3 expression at E12.5 in wild type and Olig2 null mutant embryos
(A–F) For orientation, see area indicated by the dashed box in Figure 1E. (A–C) Low resolution and (D–F) high resolution confocal images showing combined ISH for Nkx6.3 (pseudocolored red, cytoplasmic) with IHC for Hb9, Nkx6.1 and Ki67 (green, nuclear). Note that the motor neuron marker Hb9 and the proliferation marker Ki67 are not co-expressed in Nkx6.3+ cells. Nkx6.1 is co-expressed with Nkx6.3+ cells at E12.5. (G, H) Expression of Nkx6.3 is expanded in Olig2−/− mutant compared to wild type control. Scale bars: A–C, G–H 25 µM. Original magnification x40. D–F 10 µM original magnification x80.
Figure 3
Figure 3. Nkx6.3 function is dispensable for V2a interneuron development
Analysis of expression of Olig2 and Nkx6.1 (A–D); Chx10 and Gata3 (E–H); and Chx10 and Hb9 (I–L) in the caudal hindbrain of heterozygote (A, B, E, F, I, J) and Nkx6.3-null (C, D, G, H, K, L) mouse embryos at E14.5. (M) Quantification of numbers of Chx10+, Hb9+, Gata3+ and Nkx6.1+ cells per section in heterozygote (n=10) and Nkx6.3-null (n=5) embryos. Error bars indicate SEM. (B, D, F, H, J, L) are high resolution confocal photomicrographs from adjacent (A, C, E, G, I, K) photomicrographs. Scale bars: A, C, E, G, I, K 25 µM. B, D, F, H, J, L 10 µM. Original magnification A, C, E, G, I, K x40. B, D, F, H, J, L x80.
Figure 4
Figure 4. Nkx6.3 is sufficient to induce specification of V2 interneurons in the chick CNS
Chick embryos were electroporated unilaterally with the pCIG-Nkx6.3 plasmid at HH stages 9–11, and collected 44–54 h later. Whole mount embryo (left) indicates hindbrain and spinal cord A–P levels at which the analysis was performed. (A–D, M–P) GFP expression is readily detected on the electroporated side of the neural tube in green; the contralateral side lacks GFP signals and provides an internal control. Nuclei are stained blue with DAPI. Unlike the empty pCIG vector (data not shown), Nkx6.3 misexpression induced Chx10+ Lhx3+ V2 interneurons in the chick hindbrain (A, D, E, H, I, L) and spinal cord (M, P, Q, T, U, X) (areas marked by dotted circle). (I, L, U) are high resolution confocal photomicrographs showing overlap of GFP signal with Chx10, and Lhx3. Nkx6.3 misexpression further repressed appearance of Hb9+ motor neurons in the chick spinal cord (O, S, W) and hindbrain (C, G, K). (K, W) High resolution confocal photomicrographs showing lack of overlap of GFP signal with Hb9. Olig2+ pMN progenitors were also repressed in the spinal cord (N, R, V) and hindbrain (B, F, J) (areas marked by dotted circle). Forced Nkx6.3 expression did not alter the domain of Pax6 expression, which served as an internal control, as Pax6 is known to be upstream of Nkx6 factors (data not shown). Results are representative of at least 5 embyos. Scale bars: A–H and M–T 25 µM. Original magnification x40.
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