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First published online 16 May 2007
doi: 10.1242/dev.003814

Development 134, 2237-2249 (2007)
Published by The Company of Biologists 2007
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Hand2 is necessary for terminal differentiation of enteric neurons from crest-derived precursors but not for their migration into the gut or for formation of glia

Fabien D'Autréaux1, Yuka Morikawa2, Peter Cserjesi2 and Michael D. Gershon1,*

1 Department of Pathology and Cell Biology, Columbia University, P&S, New York, NY 10032, USA.
2 Department of Cell and Molecular Biology, Tulane University, New Orleans, LA 70118, USA.


Figure 1
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  		Fig. 1. Hand genes are expressed and developmentally regulated in the gut. (A) Transcripts encoding Hand2 (red), Hand1 (green), Phox2b (blue) and Mash1 (purple) quantified relative to ß-actin and plotted as a function of age. (B,C) ß-galactosidase expressed under the control of the Hand1 promoter in transgenic mice to locate sites of Hand1 expression. ß-galactosidase immunoreactivity visualized by confocal microscopy (green). Hu (B; red) or Kit (C; red) immunoreactivities co-localized with that of ß-galactosidase to identify, respectively, neurons and interstitial cells of Cajal. (D) In situ hybridization reveals Hand1 transcripts in muscle layers, but not ganglia, of adult mouse intestine, whereas transcripts encoding Hand2 are located in neurons within ganglia, but not smooth muscle. (E) Transcripts encoding Hand1, but not those encoding p75NTR, are detected, by reverse transcriptase (RT)-PCR, in a preparation from which crest-derived (CD) cells have been removed by immunoselection (nCD). Transcripts encoding Hand1 cannot be detected in a p75NTR-rich immunoselected preparation of crest-derived cells. (F) Transcripts encoding Hand2 are detected by RT-PCR in mucosa-free preparations of bowel wall (ext wall) but not in isolated mucosa from adult mouse duodenum (d), ileum-jejunum (i) and colon (c). (G,H) Transcripts encoding Hand2 were found in Hu+ (G; green) and B-FABP+ (H; red) cells. Arrows in G show Hand2 transcripts in cells that are not Hu+. d, duodenum; c, colon; CD, crest-derived; CM, circular muscle; G, ganglion; i, ileum-jejunum; LM, longitudinal muscle; M, size markers; nCD, non-crest-derived; pl, plasmid carrying DNA encoding Hand2 (positive control). Scale bars: 50 µm.

 

Figure 2
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  		Fig. 2. Hand2 immunoreactivity is intranuclear in developing enteric neurons but cytoplasmic when they mature. (A) Polyclonal antibodies raised against a 15-amino acid N-terminal sequence of Hand2 react with transfected Hand2-expressing (Hela H2) but not non-transfected Hela [Hela wild type (WT)] cells. (B) At E12, Hand2 antibodies react with crest-derived cells in the gut and prevertebral sympathetic ganglia of wild-type (WT) but not Wnt1-Cre-H2{Delta} fetuses. (C) Fetal gut dissociated at E11.5 and cultured overnight; the nuclei of Hu+ and PGP9.5+ neurons are Hand2+ (H2; red). (D) Fetal gut dissociated at E14 and cultured similarly to those in C; nuclei and cytoplasm are both Hand2+ (red). DNA is blue. (E) A dissected laminar preparation of adult bowel containing a submucosal ganglion viewed as a whole mount. Hand2 immunoreactivity (red) is restricted to the cytoplasm of Hu+ (green) neurons. DNA (blue) stained with bisbenzimide. Scale bars: 50 µm in A,B; and 25 µm in C-E.

 

Figure 3
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  		Fig. 3. Development of neurons, but not glia, in explants of fetal gut is Hand2-dependent. (A) Transcripts encoding Phox2b detected by reverse transcriptase (RT)-PCR in fetal mouse gut at E9.5 in wild-type (+/+), Hand2+/- and Hand2-/- mice. (B) Sox10+, Phox2b+ and Ret+ cells are detected at E9.5 in the outer mesenchyme of wild-type (H2+/+) and Hand2-/- (H2-/-) gut. (C) p75NTR+ cells are found in longitudinal sections cut through the foregut of wild-type and Hand2-/- mice. (D) Hu+, PGP9.5+ and NSE+ neurons develop in cultures of wild-type gut explanted at E9.5. (E) B-FABP+ (red), as well as Hu+ (green), neurons develop in cultures of wild-type gut explanted at E9.5. B-FABP+ glia, but no Hu+ neurons, develop in cultures of Hand2-/- gut explanted at E9.5. (F-H) Sox10 (F), Phox2b (G) and Ret (H) immunoreactivities are found in cultured explants of E9.5 gut from Hand2-/- and wild-type mice. (I) p75NTR+ precursors (red) are present in explants of E9.5 gut from wild-type and Hand2-/- mice; however, Hu+ neurons (green) develop in vitro only in the explants of wild-type, and not Hand2-/-, gut. (J) Co-culture of E9.5 Hand2-/- gut with somites containing primordial dorsal root ganglia. Hu+ neurons (green) develop. (K) Differentiation of neurons occurs when Hand2-/- explants are rescued by transfection with Hand2; neurons co-express Hu (green) and Hand2 (red) immunoreactivity. Ctrl, control. Scale bars: 50 µm in B-J; 10 µm in K.

 

Figure 4
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  		Fig. 4. Silencing of Hand2 prevents the in vitro development of enteric neurons. (A) Crest-derived cells immunoselected from E13 mouse gut, cultured and transfected with siRNAHand2/GFP or siRNAscrambled/GFP. siRNAHand2/GFP interfered with the development of Hu+ neurons (pink). Notice the GFP-fluorescent axons (green) of the Hu+ neurons in cultures transfected with siRNAscrambled/GFP (A; left), which are absent in those transfected with siRNAHand2/GFP (A; right). (B) Quantitative results. Overall density both of Hu+ neurons and transfected cells developing as neurons (doubly labeled with GFP and Hu) in cultures transfected with siRNAHand2/GFP are lower than those seen in cultures transfected with siRNAscrambled/GFP. siRNAHand2/GFP does not differ from siRNAscrambled/GFP in transfection efficiency measured on non-neuronal cells. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Hand2 deletion does not prevent the colonization of the bowel by enteric nervous system precursor cells in vivo but interferes with their development as neurons. (A-C) Sox10+ (green, A), Phox2b+ (red, A) and p75NTR+ (green, B) cells are present and similarly distributed in the stomachs and small intestines of E12 wild-type (WT) and Wnt-1-CreH2{Delta} mice; (C) quantified data. (D) p75NTR+ (green) and Hu+ (red) cells in the stomachs of Wnt-1-CreH2{Delta} mice (right) and wild-type littermates (left). p75NTR is present, but Hu is virtually absent, in Wnt-1-CreH2{Delta} mice. (E) The densities of Hu+ cells at E12 in the stomachs and small intestines of Wnt1-CreH2{Delta} are lower than those in the stomachs and small intestines of wild-type mice. (F) The densities of Hu+ cells in the neural tubes and dorsal root ganglion (DRG) of E12 Wnt-1-CreH2{Delta} mice and wild-type mice are similar. ns, not significant. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Terminal differentiation of enteric neurons, but not glia, is abnormal in Wnt-1-CreH2{Delta} mice. (A) nNOS+ (red) and Dbh+ (green) neurons are found in the presumptive enteric nervous system (ENS) of E12 wild-type (Wt) mice but not in Wnt-1-CreH2{Delta} bowel. (B) Hu+ (red) neurons and B-FABP+ (green) glia are present in the stomachs of wild-type mice at E12. Wnt1-CreH2{Delta} stomachs contain B-FABP+ glia but almost no Hu+ neurons. (C) The densities of B-FABP+ cells in Wnt1-CreH2{Delta} and wild-type mice are similar. (D) The number of gut cells revealed by the TUNEL technique to be undergoing apoptosis is similar in Wnt1-CreH2{Delta} and wild-type mice. The dorsal root ganglion (DRG) and neural tube were studied as positive controls. DRG, dorsal root ganglion; g, gut; L, liver; ns, not significant. Scale bars: 50 µm.

 

Figure 7
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  		Fig. 7. Hand2 is not required for precursor cells to begin neuronal differentiation. (A-F) ß3-tubulin+, GAP-43+ and PGP9.5+ neuronal precursors are present, but at a lower density, in the Wnt-1-CreH2{Delta} gut than in wild type. (A) ß3-tubulin immunoreactivity; (B) ß3-tubulin cell density; (C) GAP-43 immunoreactivity; (D) GAP-43-cell density; (E) PGP9.5 immunoreactivity; (F) PGP9.5 cell density. (G) GAP-43 (green) and PGP9.5 (red) immunoreactivities are fully coincident in wild-type (WT) mice (top) but, in Wnt-1-CreH2{Delta} animals, many PGP9.5+ cells lack GAP-43 (bottom). (H) Neural precursors lose Sox10 (green) immunoreactivity while retaining that of Phox2b (red) in both wild-type fetuses (top) and Wnt-1-CreH2{Delta} mice (bottom). Scale bars: 50 µm.

 




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