PMC

Bmp signaling was not required for Hoxb1-induced HNK-1 and Snail2 upregulation but was required for maintenance of Snail2 upregulation and repression of dorsal cell fates. Immunofluorescent detection of HNK-1 (A–C), Snail2 (D–I), Pax7 (J–L), and Pax3 (M–O) on transverse sections of neural tubes 24 ([A–F], [J–O]) and 48h PE (G–I) with Hoxb1 and noggin. Hoxb1 induced expression of HNK-1 ([A–C], asterisks in [B]) and Snail2 ([D–F], asterisks in [E]) in the presence of noggin at 24h PE but failed to maintain Snail2 expression at 48h PE (G–I). Hoxb1 no longer repressed Pax7 (J–L) and Pax3 (M–O) in the presence of noggin. Abbreviations: HNK-1, human natural killer-1; PE, postelectroporation.

Hoxb1⁺ cells generated neuronal cells, glia progenitors, and melanocytes after delamination. Immunofluorescent detection of Hoxb1 (A–M), Tuj1 (A–E), P0 (F–I), and MelEM (J–M) on transverse sections of neural tubes 72h PE of Hoxb1. Boxed areas in (C), (H), (L) are shown in (D, E), (I), and (M), respectively, in higher magnification. Hoxb1⁺ cells generated neuronal cells (arrows in [C–E]), glial progenitors (arrowheads in [H, I]), and melanocytes (arrowheads in [L, M]). Tuj1⁺ and P0⁺ cells were seen also delaminating inside the lumen of the neural tube (boxed areas in [C, H]), whereas Hoxb1⁺/P0⁺ and Hoxb1⁺/MelEm⁺ cells migrated contralaterally as well. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; PE, postelectroporation.

Expression of Hoxb1 unregulated HNK-1 and reduced neuronal differentiation. Immunohistochemical detection of HNK-1 (A–I), Hoxb1 (J–L), and Tuj1 (M–O) was performed on transverse sections of neural tubes 12h PE (A–C), 24h PE (D–F), and 48h PE (G–O) with Hoxb1. Hoxb1 upregulated HNK-1 ([A–I], asterisks in [B, E, H]) in a cell autonomous manner. Conversely, it strongly repressed neuronal differentiation as seen by Tuj1 immunofluorescence at 48h PE ([M–O], arrowheads in [N]). Electroporated cells delaminating ectopically could be seen as early as 24h PE (arrows in [D, E]) and then migrating away from the neural tube at 48h PE (arrows in [G, H, J, K, M, N]). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; HNK-1, human natural killer-1; PE, postelectroporation.

Expression of Hoxa2 in the trunk neural tube induced a neuronal progenitor to neural crest cell fate switch and limited epithelial-to-mesenchymal transition. Immunofluorescent detection of HNK-1 ([A–C], [G–I]), Hoxa2 (D–F), Tuj1 (J–L), Pax7 (M–O), Pax3 (P–R), Msx1/2 (S–U), and cadherin 6B (V–X) was performed on transverse sections of neural tubes 24h ([A–F], [M–X]) and 48h PE (G–L) with Hoxa2. Hoxa2 cell autonomously upregulated HNK-1 ([A–C] and [G–I], asterisks in [B] and [H]). Hoxa2⁺ cells delaminated 24h PE ([D–F], arrows in [D, E]). Hoxa2 strongly repressed neuronal differentiation 48h PE ([J–L], arrowheads in [K]). Hoxa2 cell autonomously repressed Pax7 ([M–O], arrowheads in [N]), Pax3 ([P–R], arrowheads in [Q]) and cadherin 6B ([V–X], arrowheads in [W]), whereas it upregulated Msx1/2 ([S–U], asterisks in [T]). Abbreviations: GFP, green fluorescent protein; HNK-1, human natural killer-1; PE, postelectroporation.

Expression of Hoxb1 represses the expression of DV markers. Detection of Pax3 ([D–F], [J–L]), Pax7 ([A–C], [G–I]), Nkx6-1 (M–O), Isl1 (P–R), Pax2 (AG–AI), and MNR2 (AJ–AL) by immunofluorescence as well as detection of Bmp4 (M–O), Bmp7 (P–R), Wnt1 (S–U), and Wnt3A (V–X) expression by in situ hybridization on transverse sections of neural tubes 12h PE (A–F), 24h PE ([G–O], [S–AF]), and 48h PE ([P–R], [AG–AL]). Expression of Hoxb1 represses expression of the dorsal markers Pax7 (arrowheads in [B], [H]) and Pax3 (arrowheads in [K]) at 12 (A–C) and 24h PE (G–I), respectively. Consistent with a cell fate switch cells electroporated with Hoxb1 cease to express the dorsal signaling molecules Bmp4 (arrowhead in [T]), Bmp7 (arrowheads in [W]), Wnt1 (arrowheads in [AB]), and Wnt3A (arrowheads in AE). Expression of Hoxb1 also represses expression of the medial marker Pax2 ([AG–AI], arrowheads in [AH]) and the ventral markers Nkx6-1 ([M–O], arrowhead in [N]), Isl1 ([P–R], arrowheads in [Q]) and NMR2 ([AJ–AL], arrowheads in [AK]) at 24h PE (M–O) and 48h PE ([P–R], [AG–AL]). Abbreviations: GFP, green fluorescent protein; PE, postelectroporation.

Optimal levels of Notch signaling were required for Hoxb1-mediated induction of neural crest cell fates and Hoxb1 itself modulates Notch signaling. Immunofluorescent detection of HNK-1 ([A–C], [J–L]), Snail2 ([D–F], [M–O]), and Msx1/2 ([G–I], [P–R]) on transverse sections of neural tubes, luciferase assays for CSL transcriptional activity (S) and Hes5 detection by in situ hybridization (T–V). All assays were done 24h PE of Hoxb1 in the presence of DAPT (A–I), of Hoxb1 with NICD ([J–R], [V]), of Hoxb1 alone (S, T), or NICD alone (U). Hoxb1 in the presence of DAPT-activated HNK-1 only at dorsal levels ([A–C], asterisks in [B]), failed to activate Snail2 (D–F) but reactivated Msx1/2 at dorsal levels ([G–I], asterisks in [H]). Hoxb1 in the presence of NICD failed to activate HNK-1 (J–L), reactivated Snail2 only marginally at dorsal levels ([M–O], asterisk in [N]) and repressed Msx1/2 ([P–R], arrowheads in [Q]). Hoxb1 electroporation increased CSL transcriptional activity (S) (p < .001). Hoxb1 on its own repressed Hes5 (arrowheads in [T]) in contrast to NICD that activated it (asterisks in [U]). Coelectroporation of Hoxb1 and NICD restored normal levels of Hes5 expression (V). Abbreviations: DAPT, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine τ-butyl ester; GFP, green fluorescent protein; HNK-1, human natural killer-1; NICD, Notch intracellular domain; PE, postelectroporation; RLUs, relative light units.

Expression of Hoxb1 induced Msx1/2 expression in embryonic stem-derived neural stem cells and induced sustained expression of Snail2 and Msx1/2 and altered cadherin expression in the chick trunk neural tube. Immunofluorescent detection of Msx1/2 (A–D) showed that its expression was slightly enhanced in Hoxb1⁻ cells (compare [A] with [B]) but dramatically upregulated in Hoxb1⁺ cells after induction of dorsal cell fates (compare [C] with [D]). Immunofluorescent detection of Snail2 (F–K), Msx1/2 (L–N) cadherin 6B (O–Q), N-cadherin (R–T), and cadherin 7 (U–W) on transverse sections of neural tubes 6h PE (F–H), 24h PE (L–W), and 48h PE (I–K) with Hoxb1. Hoxb1 upregulated Snail2 expression in a cell autonomous manner as early as 6h PE (asterisks in [G]) and maintained it at least until 48h PE (asterisks in [J]). Delaminating Snail2⁺ cells were seen at medial levels (arrow at [J]). Hoxb1 expression induced cell autonomously Msx1/2 upregulation 24h PE (arrows in [M]). It also repressed apical expression of cadherin 6B (arrowheads in [P]) and N-cadherin (arrowheads in [S]) at dorsal levels of the neural tube, whereas it induced strong upregulation of cadherin 7 (asterisks in [V]). Scale bar = 50 μm (B). Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; PE, postelectroporation.