nkx2.2b expression during early zebrafish embryogenesis. (A) Onset of nkx2.2b expression at 70% epiboly. Arrowhead points to nkx2.2b positive cells in the ectoderm. (B) Higher magnification of (A). (C) Dosal view of (B). (D) nkx2.2b expression in the developing ventral head at bud stage. (E-G) Lateral view of prospective head regions of embryos at bud stage (anterior to left, dorsal to top) with expression of nkx2.2b (E), shh (F) and flh (G) in bright-field (E,G) and under fluorescence light (F). The asterisk indicates the anterior border of the prechordal plate. Note overlap of nkx2.2b with shh and flh expression in the anterior mesoderm. (H) nkx2.2a expression precedes that of nkx2.2b and is expressed in head mesoderm at 80% epiboly. (I-L) Shh precedes posterior extension of nkx2.2b expression in the ventral neural tube. (I) Expression of shh in notochord and MFP at the 5s stage. Arrowhead indicates posterior front of shh expression in the MFP. (J-L) nkx2.2b expression at 5s (J), 7s (K) and 9s (L) stages. Arrowheads in (J-L) show the posterior front of extending nkx2.2b expression in the LFP. All pictures, except C, are lateral views with anterior to the left; dashed lines indicate the boarder between ectoderm and mesoderm (B,G). Scale bar in (D) represents 100 µm (for A,D), in (H) 30 µm (for B,C,E-H), and in (L) 50 µm (for I-L).

Differences in the expression patterns of nkx2.2a and nkx2.2b. nkx2.2a (A) and nkx2.2b (B) expression in embryos at the 18s stage. (C-F) Higher magnification of rostral (C,D) and caudal (E,F) head regions showing nkx2.2a (C,E) and nkx2.2b (E,F) expression in the ventral brain. Note differences in expression in the mid-diencephalic boundary (MDB), the mid-hindbrain boundary (MHB) and in the pancreatic progenitor region (pc). (G-J) Transversal sections showing expression of nkx2.2a (G,I) and nkx2.2b (H,J) in the ventral CNS at hindbrain (G,H) and trunk (I,J) levels (positions of sections indicated in A,B). (K,L) Gradual reduction of nkx2.2a (K), but maintenance of strong nkx2.2b expression (L) in the posterior trunk. (M-S) Transversal sections through the spinal cord of 18s wild-type embryos showing expression of foxa2 (M), nkx2.2b (blue) and shh (red) (N), tal2 (O), olig2 (red) and nkx2.2b (blue) (P), isl1 (Q), isl1 and nkx2.2b (R) at mid-trunk level, and pax6 at posterior trunk level (S). LFP, lateral floor plate; MDB, mid-diencephalic boundary; MFP, medial floor plate; MHB, midbrain-hindbrain boundary; mn, motoneurons; nc, notochord; pc, pancreatic progenitor cells; sn, sensory neurons. (A-F) lateral views and (K,L) dorsal views, anterior to the left. Scale bar in (B) represents 200 µm (for A,B), in (D) 100 µm (for C,D), in (F) 50 µm (for E,F), in (L) 30 µm (for K,L), and in (S) 20 µm (for G,I,H,J,M-S).

Ectopic shh, but not cyc expression results in dorsal expansion of nkx2.2b. (A-F) Expression of nkx2.2b in 18s wild-type embryos (A-C) and embryos injected with 2 pg cyc RNA (D-F). Note enlargement of axial structures (notochord, MFP) evident in transversal section in (F), but only mildly affected nkx2.2b expression in head (D) and trunk (E,F). (G-I) Expression of nkx2.2b in oep-/- mutants. Note missing MFP (I), but normal nkx2.2b expression in head (G) and trunk (H,I). (J-L) nkx2.2b expression in embryos injected with 80 pg shh RNA. nkx2.2b shows strong dorsal expansion in distinct regions of the brain and the complete spinal cord. (A,D,G,J) are lateral views of the head and (B,E,H,K) of the trunk, anterior to the left. (C,F,I,L) are transversal sections through the neural tube at trunk levels. Scale bar in (J) represents 100 µm (for A,D,G,J), in (K) 50 µm (for B,E,H,K), and in (L) 20 µm (for C,F,I,L).

Differential regulation of nkx2.2b expression in hedgehog mutants. nkx2.2b expression in wild-type (A,B), yot (C,D), dtr (E,F), syu (G,H) and smu (I,J) 18s stage embryos. Left panel is lateral overview of embryos, right panels are dorsal views of trunk region. Small insets in left panel show lateral views of wild-type siblings from same clutch of heterozygous carriers. Note gradual loss of nkx2.2b expression in head regions of homozygous mutants. Arrowheads indicate areas with loss of nkx2.2b expression. Scale bar in (I) represents 200 µm (for A,C,E,G,I) and in (J) 30 µm (for B,D,F,H,J).

Isl1 and retinoids repress nkx2.2b expression. (A-D) Retinoic acid (RA) treatment blocks nkx2.2b expression. (A,B) Lateral views of 14s control embryo incubated in 10% DMSO showing nkx2.2b, as well as emx1/krox20 (inset in A) and olig2 (inset in B) expression. (C,D) Treatment with 100 nM (C) and 100 µM all-trans RA (D), results in a strong downregulation of nkx2.2b expression in the head (C) and a complete block of nkx2.2b in the trunk (D). Note rostral expansion of krox20 in rhombomere 5 (inset in C) and up-regulated olig2 expression in the trunk (inset in D) indicative for RA induced posteriorization, as described previously (Begemann and Meyer, 2001). (E-J) Interference with endogenous retinoid synthesis by DEAB treatment up-regulates nkx2.2b. (E-G) Expression of nkx2.2b in head (E) and trunk (transversal section in F), emx1/krox20 in head (inset in E) and olig2 in the trunk (transversal section in G) of control embryos treated with 10% DMSO. (H-J) Treatment of embryos with 10 µM DEAB results in the expansion of nkx2.2b expression in the head (H) and trunk (I) and a reduction of olig2 (J). Change of krox20 expression (inset in H) confirms anteriorization as described previously (Begemann et al., 2004). (K-Q) Ectopic isl1 expression inhibits nkx2.2b activation in the trunk. Expression of nkx2.2b (K,N), foxa2 (L,O,P) and Znp-1 (M,Q) in a non-injected control embryo (K-M) and in an embryo injected with 70 pg isl1 RNA at the 18s stage (N-Q). In isl1 injected embryos, foxa2 is reduced to the MFP (O) or completely lost (N,P). In some areas, the ventral most neural tube cells express Znp-1 (arrow in Q). (R) Schematic model of interactions in the ventral neural tube restricting nkx2.2b expression to the LFP. RA, signalling induced by retinoids derived from paraxial mesoderm. Scale bar in (H) represents 100 µm (for A-H), in (J and Q) 20 µm (for F,G,I,J,L,M,O-Q), and in (N) 100 µm (for K,N).