Sonic hedgehog rescues cranial neural crest from cell death induced by ethanol exposure Sara C. Ahlgren*, Vijaya Thakur, and Marianne Bronner-Fraser Division of Biology 139-74, California Institute of Technology, Pasadena, CA 91125 Communicated by Eric H. Davidson, California Institute of Technology, Pasadena, CA, June 14, 2002 (received for review March 14, 2002) Alcohol is a teratogen that induces a variety of abnormalities Materials and Methods including brain and facial defects [Jones, K. & Smith, D. (1973) Embryos. Chicken eggs were obtained from local sources. Eggs Lancet 2, 999-1001], with the exact nature of the deficit depending were incubated at 37°C with constant humidity. In preliminary on the time and magnitude of the dose of ethanol to which experiments, unopened eggs were injected with 200 ␮lofa developing fetuses are exposed. In addition to abnormal facial solution of 10% ethanol in Ringer’s solution, or with Ringer’s structures, ethanol-treated embryos exhibit a highly characteristic solution alone, at 26 h of incubation. This procedure has been pattern of cell death. Dying cells are observed in the premigratory previously demonstrated to result in craniofacial abnormalities and migratory neural crest cells that normally populate most facial (6). We then modified this procedure, such that eggs were structures. The observation that blocking Sonic hedgehog (Shh) opened at stage 9–10, and 20 ␮l of a 1% ethanol solution (in signaling results in similar craniofacial abnormalities prompted us Ringers) was placed between the vitelline membrane and the to examine whether there was a link between this aspect of fetal embryo. Both procedures resulted in similar cranial neural crest alcohol syndrome and loss of Shh. We demonstrate that adminis- cell death and loss of frontonasal tissues. Embryos were col- tration of ethanol to chick embryos results in a dramatic loss of Shh, lected at the desired time, and either harvested in TRIzol (Life as well as a loss of transcripts involved in Shh signaling pathways. Technologies, Grand Island, NY) for RNA isolation or fixed in In contrast, other signaling molecules examined do not demon- 4% paraformaldehyde (PFA) for either cell death analysis or in strate such dramatic changes. Furthermore, we demonstrate that situ hybridization. Whole-mount in situ hybridizations were both the ethanol-induced cranial neural crest cell death and the performed as described (11). Whole mount HNK-1 staining was associated craniofacial growth defect can be rescued by application performed by soaking fixed embryos in HNK-1 supernatant of Shh. These data suggest that craniofacial anomalies resulting (Development Studies Hybridoma Bank, Iowa City), followed by from fetal alcohol exposure are caused at least partially by loss of overnight incubation in 1:100 goat anti-mouse IgM–AP Shh and subsequent neural crest cell death. (Zymed), using BCIP (5-bromo-4-chloro-3-indolyl phosphate)͞ NBT (nitroblue tetrazolium chloride) to visualize. he secreted protein Sonic hedgehog (Shh) is essential for Tmultiple events during embryogenesis, including proper Cranial Neural Crest Cell Death. Fixed embryos were cryoprotected craniofacial development (1–4). Of the vertebrate homologues in sucrose, then embedded in gelatin and sectioned on a cryostat of the Drosophilia segment polarity gene Hedgehog, Shh has the at 10 ␮m. Sections were briefly heated to 42°C to remove the widest expression pattern in the developing head, including the gelatin, then blocked in 20% heat-inactivated goat serum in a presence of transcripts in the developing branchial arches during solution of PBSϩBSA ϩ 0.1% Triton, with all further dilutions the time of neural crest migration (5). SHH mutations in both also being made in this solution. The sections were incubated mice and humans cause significant defects in head development, overnight at 4°C in HNK-1 supernatant (1:10), followed by including distinctive facial and brain abnormalities (3, 4). Anal- anti-mouse IgM–FITC (1:500; Zymed). Finally, the sections ogously, inhibition of Shh-signaling during facial development were incubated for 10 min in 10 ␮g͞ml of the nuclear marker causes neural crest cell death and a subsequent reduction in head 4Ј,6-diamidino-2-phenylindole (DAPI) made in water, then cov- size (1). erslips were applied. The percentage of dead cranial crest cells There are several intriguing similarities in the craniofacial was determined as described (1). All statistics were performed phenotypes observed in embryos treated with ethanol and those using STATVIEW software. treated with antibodies that block Shh signaling. Both treatments result in a reduction in head size and selective apoptosis of Size Measurements. Embryos were photographed at fixed mag- cranial neural crest cells. The neural crest cell death observed nifications, which changed at different ages and with different after blocking Shh signaling in the chick (1) is similar to that head positions. The digital photographs were analyzed using the observed after ethanol exposure in a number of species including measuring tool available in PHOTOSHOP software (Adobe Sys- mouse, chick, and quail, both in vivo and in vitro (6–10). tems, Mountain View, CA). However, the mechanisms by which alcohol affects selective target tissues remain unknown. Hybridoma Treatments. Stage 9–10 chick embryos were injected In this study, we investigate the impact of embryonic exposure with a function-blocking anti-Shh antibody (Development Stud- to ethanol on Shh signaling during the critical period of early ies Hybridoma Bank) as described (1). cranial neural crest migration. After ethanol exposure, we noted a reduction in the mRNA levels of Shh both by in situ hybrid- RT-PCR. RNA was pooled from 4–6 embryos per experiment and ization and reverse transcriptase (RT)-PCR, as well as a reduc- treated to remove genomic DNA, then cDNA was synthesized tion in the mRNA for a number of genes in the Shh signaling using SuperscriptII. Alternatively, RNA was collected from the cascade. We further demonstrate that cranial neural crest cells heads of 6–7 embryos per condition. The relative amounts of can be rescued from ethanol-induced cell death, and that the cDNA in each matched set was normalized for the presence of associated decrease in size of the frontonasal mass can be prevented by application of exogenous Shh. We have thus identified a molecular deficit associated with embryonic expo- Abbreviations: Shh, Sonic hedgehog; RT, reverse transcriptase; DAPI, 4Ј,6-diamidino-2- sure to ethanol that in turn represents a link to the etiology of phenylindole. craniofacial defects. *To whom reprint requests should be addressed. E-mail: [email protected]. 10476–10481 ͉ PNAS ͉ August 6, 2002 ͉ vol. 99 ͉ no. 16 www.pnas.org͞cgi͞doi͞10.1073͞pnas.162356199 Downloaded by guest on October 2, 2021 Fig. 2. There is increased cranial neural crest cell death after ethanol Fig. 1. Ethanol treatment results in a reduction in the size of craniofacial tissues. treatment. Embryos were treated with ethanol at stage 9–10, collected 1 day (a and b) Embryos collected approximately 24 h after ethanol treatment (stage later, fixed, sectioned, and the sections stained for the neural crest marker 9–10) show distinct differences in craniofacial growth. The measurements for the HNK-1 (in green) and the nuclear marker DAPI (in blue). (a and b) A section frontonasal mass are indicated by the bar in each figure; control, 0.42 mm; taken from the optic cup (oc) region of a control embryo contains HNK-1- ethanol, 0.33 mm (see Table 1). (c–f) HNK-1 whole-mount analysis of embryos Ϸ1 positive neural crest cells (nc). (c and d) A section taken from the same region day after ethanol treatment demonstrates a reduction in the HNK-1-positive of an ethanol-treated embryo also contains HNK-1-positive neural crest cells. cranial neural crest. Streams of neural crest are indicated with arrows, and the ec, ectoderm; di, diencephalon. (e–eЈЈ). Magnified views of the area outlined region around the otic vesicle (ov) is enlarged in d and e. Although crest is clearly in a. A single pyknotic nucleus is present (arrow). This nucleus has an HNK-1- present in all appropriate regions, the intensity of staining appears reduced in the positive membrane (eЈ and eЈЈ). (f–fЈЈ). Magnified views of the area outlined whole-mount. HNK-1 staining of non-neural tissue, including the heart (h) ap- in c. Arrows point to three examples of pyknotic nuclei in f, all of which have pears unchanged. (f) The trunk region of ethanol-treated embryos did not HNK-1-positive membranes (fЈ). (g) Analysis of the pyknotic cells demonstrates display any neural crest abnormalities when stained with HNK-1. (g) Ethanol- that exposure to ethanol resulted in a dramatic increase in the percentage of treated (stage 9–10) embryos collected 3 days after treatment show distinct size the cranial neural crest cells (HNK-1-positive) that appear pyknotic, whereas differences of the frontonasal mass (Table 1). no change in the survival of the non-neural crest mesenchyme (HNK-1- negative) was observed. HNK-1-positive: control n ϭ 44; ethanol n ϭ 48. *, Unpaired t test, P Ͻ 0.0001. HNK-1-negative: control n ϭ 44; ethanol n ϭ 39, the ubiquitous gene GAPDH (12), using a Molecular Dynamics P not significant. (h) Merged image of DAPI and HNK-1 from the region PhosphorImager and IMAGEQUANT software (Molecular Dy- marked ‘‘h’’ in c, a region that contains almost no neural crest cells, and namics). GAPDH was used as a loading control for each PCR demonstrates no pyknotic nuclei. reaction. By the amounts of GAPDH and by visual assessment of 18S RNA, there did not appear to be a significant alteration in cShh, cFgf-8, or control constructs. The resulting supernatant the amount of RNA that was obtained from ethanol-treated versus control embryos.