Effects of calcium and estrogen on the development of the ceratohyal cartilage in zebrafish (Danio rerio) larvae upon embryo and maternal cadmium exposure.

The present study is to investigate the reason why the ceratohyal cartilage (CH) angle of zebrafish larvae were larger compared to the control group after their female parents were treated with cadmium (F-Cd). However, the CH angle was smaller compared to the control group when embryos were directly exposed to Cd2+ for 72 h (D-Cd). Results showed that calcium contents of larvae were lower than the control, but the transporter isoforms trpv4 and trpv6 mRNA expressions were significantly increased upon D-Cd treatment. Furthermore, external Ca2+ added during D-Cd treatment reveals that the CH angles of larvae did not appear significantly different compared to the control. On the other hand, E2 (17ß-estradiol) contents were higher around 1.9 folds in the ovaries of females; CH angle were over 25°, and Cd2+ contents were higher around 6 folds than the control group on larvae treated through F-Cd treatment; CH angles and E2 levels on larvae were higher than the control after the larvae were treated with 1.84 µM E2 (D-E2); Estradiol receptor (ER) isoforms ERß1 and ERα mRNA expressions significantly increased when 0 hpf embryos were either treated with D-E2 or D-Cd. According to the results, we suggested that the CH angle of larvae become larger upon F-Cd treatment due to maternal Cd2+ inducing E2 levels. However, the CH angle of larvae appeared to be smaller compared to the control upon D-Cd treatment. We suggested that the CH angle decreased due to the decrease of Ca2+ contents upon Cd2+ exposure.

Changes in the inner ear structures in cystic fibrosis patients.

OBJECTIVE: Although prolonged use of antibiotics is very common in cystic fibrosis (CF) patients, no studies have assessed the changes in both cochlear and peripheral vestibular systems in this population. METHODS: We used human temporal bones to analyze the density of vestibular dark, transitional, and hair cells in specimens from CF patients who were exposed to several types of antibiotics, as compared with specimens from an age-matched control group with no history of ear disease or antibiotic use. Additionally, we analyzed the changes in the elements of the cochlea (hair cells, spiral ganglion neurons, and the area of the stria vascularis). Data was gathered using differential interference contrast microscopy and light microscopy. RESULTS: In the CF group, 83% of patients were exposed to some ototoxic drugs, such as aminoglycosides. As compared with the control group, the density of both type I and type II vestibular hair cells was significantly lower in all structures analyzed; the number of dark cells was significantly lower in the lateral and posterior semicircular canals. We noted a trend toward a lower number of both inner and outer cochlear hair cells at all turns of the cochlea. The number of spiral ganglion neurons in Rosenthal's canal at the apical turn of the cochlea was significantly lower; furthermore, the area of the stria vascularis at the apical turn of the cochlea was significantly smaller. CONCLUSIONS: Deterioration of cochlear and vestibular structures in CF patients might be related to their exposure to ototoxic antibiotics. Well-designed case-control studies are necessary to rule out the effect of CF itself.

Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone.

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.

Pentoxifylline-tocopherol-clodronate combination: A novel treatment for osteoradionecrosis of the temporal bone.

BACKGROUND: Osteoradionecrosis (ORN) of the temporal bone is a complication of radiation therapy that is extremely challenging to manage. METHODS: We report the case of a patient treated at our institution and present a review of the literature. RESULTS: A 52-year-old woman presented with ORN of the temporal bone 20 years after radiation therapy for an ipsilateral parotid tumor. She failed conservative management, including serial debridements, oral and topical antibiotics/antifungals, and aural lavage. As an alternative to hyperbaric oxygen (HBO) and/or temporal bone resection, treatment with pentoxifylline-tocopherol-clodronate combination (PENTOCLO) was pursued and her condition improved dramatically. CONCLUSION: This is the first document use of PENTOCLO to treat ORN of the temporal bone. PENTOCLO may represent an effective nonsurgical management option for ORN of the temporal bone.

Rusty green stained temporal bone associated with exposure to tetracycline: an unusual presentation of black bone disease.

OBJECTIVE: To review the phenomenon and implications of temporal bone and craniofacial bone staining in the context of prolonged exposure to tetracycline antibiotic. METHODS: Case report and literature review. RESULTS: A 52-year-old male with a 5-year history of tetracycline use presented to undergo tympanomastoidectomy and was found to have an unusual rusty green pigmentation of the entire aspect of the exposed temporal bone. A literature review revealed more than 20 cases of tetracycline-induced pigmentation of intraoral maxillary and mandibular bone, and 2 prior cases involving the cranial bones. CONCLUSION: Tissue and organ pigmentation is an unexpected and unfavourable consequence of the use of tetracyclines, particularly minocycline. Tetracycline is contraindicated in children because of the risk for dysosteogenesis and enamel hypoplasia. In adults, although the unusual staining may present as an unexpected dilemma upon surgical exposure, current research shows no significant clinical consequences for this type of pigmentation.

Denervation and beta2-adrenoceptor-agonist administration on craniofacial bone density.

OBJECTIVE: Beta2-agonist medications are thought to have adverse effects on bone density. Surgical denervation and intramuscular beta2-agonist injections appear to have opposing effects on skeletal muscles. The present study has been designed to assess the effects of denervation of the masseter, intramuscular injection of a beta2-agonist and the combination of both procedures, on bone density in the craniofacial skeleton in rats. MATERIALS AND METHODS: Sprague-Dawley rats were prepared as four groups: 1. surgical sham + saline injection into the masseter (sham); 2. surgical denervation of the masseter (den.); 3. surgical denervation of the masseter + intramuscular formoterol injection into the affected muscle (den.+form.); 4. intramuscular formoterol injection into the masseter (form.). All specimens were submitted for CT examination and volumetric calculations of the mineralised bone tissue were performed. RESULTS: The sham and form. groups had a greater volume of mineralised bone in the zygoma on the experimental side compared with the control side. The maxilla on the experimental side had a higher volume of mineralised bone in the den.+form. and form. groups compared with the sham and den. groups. The control side of the maxilla had a higher volume of mineralised bone in the den.+form. and form. groups compared with the den. group only. CONCLUSION: Intramuscular administration of formoterol appears to induce a bilateral increase in bone mineral density in the maxilla and the zygoma, likely explained as a secondary effect of the well-described increase in muscle mass and strength associated with beta2-agonist administration.

The effects of denervation and formoterol administration on facial growth.

OBJECTIVE: To identify and demonstrate possible alterations of skeletal structures which might follow either unilateral surgical denervation of the masseter muscle, unilateral intramuscular injection of formoterol directly into the masseter muscle, or intramuscular formoterol injection after surgical denervation. MATERIALS AND METHODS: Male Sprague Dawley rats (N = 16; four weeks of age) were prepared as four groups: 1. surgical sham + saline injection into the masseter muscle (sham); 2. surgical denervation of the masseter muscle only (den.); 3. surgical denervation of the masseter muscle plus intramuscular formoterol injection into the affected muscle (den.+form.); 4. intramuscular formoterol injection into the masseter muscle only (form.). The specimens were submitted for CT examination, the skulls and hemimandibles were photographed and measurements of craniofacial bones were made. RESULTS: In this relatively small sample, comparisons between non-experimental and experimental sides revealed differences, both within the groups and for the same measurements between groups, with the den. and den.+form. groups showing the most change. Relative increases in the gonial angle shown in these groups occurred bilaterally, with the change on the experimental side always greater in magnitude than the change on the contralateral side. CONCLUSIONS: Surgical denervation of the masseter muscle leads to an alteration in the size and shape of the skeletal structures close to the zygoma and the mandible. The intramuscular injection of formoterol into denervated masseter muscle seems to limit this skeletal alteration after surgical denervation.

Preliminary evaluation of histological changes found in a mechanical arthropatic temporomandibular joint (TMJ) exposed to an intra-articular Hyaluronic acid (HA) injection, in a rat model.

OBJECTIVE: This study investigates the histological effects of Hyaluronic acid injections in the treatment of induced temporomandibular joint (TMJ) osteoarthritis in rats. STUDY DESIGN: Twenty-four male Wister rats were subjected to induced mechanical osteoarthritis by manual hypermobility for 10 successive days. Animals were then divided into two groups; group I (control) and group II (experimental). Ten days after the induction of hypermobility, the right TMJ of the experimental animals was injected with a dose of 0.12 mg HA intra-articularly and 0.12 mg saline was injected into the left joint; while animals in the control group were left without any treatment. Two rats from group I were killed at one, two and six weeks; while 6 animals from group II were killed at one, two and four weeks post injection. RESULTS: The disk of the right joints in the experimental animals was of normal thickness and there was an increase in the thickness of the fibrocartilagenous layer. In the left joint; ulcerative changes in the disk were evident where the fibres were not well oriented and scalloped areas in the temporal bone area were present denoting osteoclastic activity. CONCLUSIONS: Repeated intra-articular TMJ injection of Hyaluronic acid appears to be a safe and effective way of inhibiting the progression of osteoarthritic changes in the joint through development of articular cartilage and reducing fibrous tissue proliferation.

Topical application of calcium channel blockers to reduce the progression of experimentally induced myringosclerosis and tympanosclerosis.

OBJECTIVES: This study aimed to evaluate the ability of topically applied calcium channel blockers (diltiazem) to reduce the progression of experimentally induced myringosclerosis and tympanosclerosis. STUDY DESIGN: Animal model. Experimental prospective study. METHODS: The study included 25 adult albino guinea pigs that were bilaterally myringotomized and inoculated with a suspension of Streptococcus pneumonia type 3. The right ears were treated with topical application of diltiazem, and the untreated left ears served as the control group. Otomicroscopy and remyringotomy were conducted every week. One animal was sacrificed after 1 week and the remaining at the end of 6 weeks. Temporal bones were dissected, and tympanic bullae were analyzed with light microscopy. RESULTS: The untreated control ears showed evidence of extensive myringosclerosis on otomicroscopy, and the ears treated with calcium channel blockers did as well although to a lesser degree. Under light microscopy, the lamina propria of both tympanic membranes and middle ear mucosae of the control group exhibited thicker (P < .1 and P < .05, respectively) and larger (P < .01 and P < .01, respectively) sclerotic tissue in comparison with the treatment group. CONCLUSION: The results suggest that calcium channel blockers had an influence in the prevention of tympanosclerosis.

Functional and morphological effects of fotemustine on the auditory system of the rat.

OBJECTIVE: This study aimed to elucidate the potential inner-ear effects of fotemustine, a chemotherapeutic agent which crosses the blood-brain barrier and is used in the treatment of primary and metastatic brain tumours and metastatic melanoma. METHODS: This study utilised distortion product otoacoustic emissions and transmission electron microscopy in order to conduct electrophysiological and morphological assessments, using a rat experimental model. Twelve ears of six male rats were examined two months following intraperitoneal slow infusion of fotemustine (100 mg/m2 or 7.4 mg/kg). Pre- and post-treatment measurements were compared. Finally, electron microscopy was performed on three rat temporal bones. RESULTS: After infusion of fotemustine, distortion product otoacoustic emissions revealed a significant reduction in signal-to-noise ratios only at 3600 Hz (from 11.95 +/- 7.52 to -0.26 +/- 9.45 dB) and at 3961 Hz (from 18.09 +/- 7.49 to 6.74 +/- 12.11 dB) (referenced to 2f1 - f2). Transmission electron microscopy of the temporal bone revealed ultrastructural changes in the outer hair cells, stria vascularis and cochlear ganglion at the cochlear basal turn. The ganglion cell perikarya were unaffected. CONCLUSIONS: Fotemustine was administered via intraperitoneal slow infusion in a rat experimental model. Twelve ears of six survivors, from 10 rats, were evaluated at the second month. Fotemustine was determined to have a potential for ototoxicity at 3600 and 3961 Hz. Three randomly chosen rats underwent electron microscopy for morphological analysis. Morphological effects in the cochlear basal turn were observed. Oedematous intracytoplasmic spaces and perivascular areas of the stria vascularis, as well as distorted chromatin content, were detected, thereby suggesting potential ototoxic effects for this agent. Further experimental and clinical studies are required in order to determine whether the effect seen in this pilot study is reversible, and to analyse effects in humans.

The effect of etodolac on experimental temporomandibular joint osteoarthritis in dogs.

PURPOSE: The purpose of this study was to test the effect of etodolac on a dog model of osteoarthritic temporomandibular joints (TMJ). MATERIAL AND METHODS: Ten adult beagle dogs underwent surgery to induce osteoarthritis in the right TMJ, and were then randomly divided into two groups each of 5 dogs. From 2 to 6 weeks after surgery, the dogs in group 1 (control group) were given an empty gelatin capsule daily, and the dogs in group 2 (study group) were given 15 mg/kg of etodolac daily. All joints were examined radiologically and histologically 8 weeks postoperatively. RESULTS: In group 1, the operated right joints showed severe osteoarthritic changes in the condyle and severe morphological differences in comparison with the unoperated (left joint). Group 2 showed fewer morphological differences between the unoperated control (left) and the operated (right) TMJ. Osteoarthritic changes in the TMJ were significantly less severe in group 2 (etodolac) than in group 1. CONCLUSION: Oral administration of etodolac at a dose of 15 mg/kg minimized the continuation of experimentally induced osteoarthritic changes in the canine TMJ, when compared with the placebo. Thus, etodolac may play a role in preventing progression of human TMJ osteoarthritis.

Unbiased quantification of Scarpa's ganglion neurons in aminoglycoside ototoxicity.

While most studies have demonstrated damage to the cochlear and vestibular endorgan as the primary site of aminoglycoside toxicity, the effect on the primary afferent neurons of the vestibular ganglion remains to be determined. This study used the unbiased stereology-optical fractionator method to obtain estimates of the vestibular ganglion neuronal number. Archival temporal bone specimens from seven subjects with a history of gentamicin (n=3) and streptomycin (n=4) aminoglycoside ototoxicity were used. The post-ototoxicity survival time ranged from two months to 8 years, with an average of 2.2 years. Seven archival human temporal bone specimens from age-matched subjects with no history of audiovestibular symptoms or ototoxicity served as controls. Group means were compared using unpaired, two-tailed student's t test. The average vestibular ganglion neuronal number in the aminoglycoside ototoxicity group was 20, 733 neurons (CV=0.073), which was significantly lower (p<0.005) than the average number in the age-matched control group of 24, 902 neurons (CV=0.109). These findings may be consistent with either retrograde degeneration or a direct neurotoxic effect of the aminoglycosides on the vestibular ganglion neuron.

Cupular deposits and aminoglycoside administration in human temporal bones.

In this study, the deposits of basophilic material on the cupula of the semicircular canals in temporal bones from patients who had aminoglycoside administration within six months prior to death were compared with normal temporal bones. Subjects were divided into two groups. Group I included 24 normal control temporal bones age-matched to group II patients. Group II consisted of 23 temporal bones that had received aminoglycosides within six months prior to death. All temporal bones were examined under light microscopy. One (4.2 per cent) of 24 temporal bones in group I (normal) showed basophilic deposits. In group II, deposits were observed in 8 (34.8 per cent) of 23 temporal bones. The prevalence of basophilic deposits in group II was significantly higher than group I. This study demonstrates that within six months after aminoglycoside administration there is an increased prevalence of basophilic deposits on the surface of the cupula. Such changes may be related to the benign paroxysmal positional vertigo (BPPV) seen in some patients who have had aminoglycoside administration.

Effects of aminoglycoside administration on cochlear elements in human temporal bones.

OBJECTIVE: Although there have been numerous reports on the relationship between the period of aminoglycoside administration and cochlear damage in animals, to date there have been no such studies in humans. The purpose of this study is to observe the early and late cochlear effects of aminoglycoside administration on hair cells, spiral ganglion cells, stria vascularis, and spiral ligament. METHODS: Specimens were divided into three groups. Group I included "normal" temporal bones with no histopathologic findings of otitis media and no history of otologic or ototoxic drug administration. Group II consisted of temporal bones that received aminoglycosides within 2 weeks before death and group III of temporal bones that had aminoglycosides from 2 weeks to 6 months prior to death. Patients in groups II and III received gentamycin, kanamycin or tobramycin. Temporal bones were excluded from groups II and III if patients had a history of otologic disease or other ototoxic drugs. All temporal bones were examined under light microscopy. Standard cytocochleograms and spiral ganglion cell reconstructions were done on all temporal bones. Morphometric measurements of areas of stria vascularis were made in all turns of the cochlea on mid-modiolar sections. Spiral ligament was divided into four segments according to the locations of different types of fibrocytes. The mean loss of fibrocytes in each segment was estimated. RESULTS: The percentages of intact outer hair cells in the basal turn were significantly greater in group I compared to groups II and III. The mean area of the stria vascularis in the apical turn was significantly less in groups II and III compared to group I. CONCLUSION: This study demonstrates that in a short period (within 2 weeks) after aminoglycoside administration, a decrease in hair cells and in the area of the stria vascularis occurred.

Effect of parenteral aminoglycoside administration on dark cells in the crista ampullaris.

OBJECTIVE: To observe the early and late effects of the parenteral administration of aminoglycosides on the dark cells of ampullae in the inner ear. STUDY DESIGN: Comparative study of the histopathologic characteristic of human temporal bones. SUBJECTS AND METHODS: Sixty-three temporal bones from 44 subjects (age range, 16-81 years) were examined by light microscopy. Three groups of temporal bones were selected for this study: group 1, 30 "normal" temporal bones from 22 subjects (mean age, 59 years; age range, 25-81 years) with no history or histopathologic findings of otologic disease or ototoxic drug use; group 2, 14 temporal bones from patients who received aminoglycoside treatment within 2 weeks before death; and group 3, 19 temporal bones from patients who received aminoglycoside treatment between 2 weeks and 6 months before death. RESULTS: The mean +/- SD number of dark cells in group 1 was 15.0 +/- 2.47; in group 2, it was 17.3 +/- 1.93 in the subjects who received gentamicin sulfate and 15.0 +/- 3.08 in those who received kanamycin sulfate and tobramycin; in group 3, it was 14.6 +/- 1.67 in the subjects who received gentamicin and 15.2 +/- 2.31 in those who received kanamycin and tobramycin. The overall difference between the 3 groups was not statistically significant (P =.07). The cytologic characteristics of dark cells were similar in all 3 groups. The number of dark cells showed a decline with increasing age in group 1. CONCLUSIONS: The result of this study suggests that the treatment period was probably too short to destroy the dark cells. Therefore, long-term aminoglycoside therapy may be necessary to get a more permanent result.

Gentamicin tympanoclysis: effects on the labyrinthine sensory cells.

OBJECTIVES: The objective of the study was to determine whether a selective vestibular hair cell toxicity with sparing of the cochlear hair cells could be achieved by infusing different concentrations of gentamicin into the middle ears of adult cats. STUDY DESIGN: Prospective experimental animal study treating only the left ear of each cat, the right ear serving as individual control. METHODS: Gentamicin solution at concentrations of either 30 or 3 mg/mL was infused daily into the left middle ear of adult cats until overt ataxia occurred. After 1 month or 6 months, each cat was killed and its temporal bones prepared for optical microscopy. RESULTS: Animals treated with 30 mg/mL gentamicin until ataxic required a median of five daily doses. These animals had clear-cut cochlear basal turn hair cell losses accompanying toxic lesions in the utricle and cristae. In contrast, animals treated with 3 mg/mL gentamicin until ataxic required an average of 19 daily doses. These animals had lesions restricted to the utricle and cristae with sparing of the cochlea hair cells. Animals that failed to develop ataxia manifested neither lesions of the cochlear nor vestibular hair cells. CONCLUSION: Gentamicin tympanoclysis in the cat animal model, using a dilute solution and continued once daily until clinical ataxia occurs, is capable of producing selective vestibular hair cell toxicity while sparing cochlea hair cells.

The effect of growth hormone therapy on mandibular and cranial base development in children treated with total body irradiation.

The aim of the present investigation was to study craniomandibular development during growth hormone (GH) therapy in nine girls and one boy, aged between 7.3 and 16 years, who exhibited pronounced growth reduction after total body irradiation (TBI) and bone marrow transplantation (BMT). Age- and sex-matched healthy children with normal dentofacial development constituted the control material. The investigation data were based on measurements made on lateral skull radiographs taken at the start and, on average, 6 months after cessation of GH treatment. The control group comprised similar longitudinal cephalographic records. The results showed that GH therapy in patients who exhibited growth retardation after TBI and BMT had only a minor effect on cranial base dimensions, probably due to the fact that the development of this area is completed at a relatively early age. The effect of GH treatment on mandibular growth was very obvious. The dimensional increase of the mandibular variables in the patients was equivalent to, or in some cases even exceeded, that of the controls. In relation to basion, the mandibular condyles were displaced in a backward/upward direction in the patient group. Displacement in the opposite direction was recorded in the controls. It seems likely that the development seen in the patients is a reflection of a normalization of the condyle-fossa relationship made possible by enhanced condylar growth. This change should be advantageous for the function of the craniomandibular complex.

Neurotoxic mechanism of cinnabar and mercuric sulfide on the vestibulo-ocular reflex system of guinea pigs.

Cinnabar, a naturally occurring mercuric sulfide (HgS), has been combined with Chinese herbal medicine as a sedative for more than 2000 years. To date, its neurotoxic effect on the vestibulo-ocular reflex (VOR) system has not been reported. By means of a caloric test coupled with electronystagmographic recordings, the effect of commercial HgS and cinnabar on the VOR system of guinea pigs was studied. HgS or cinnabar was administered orally (1.0 g/kg) to Hartley-strain guinea pigs once daily for 7 consecutive days. A battery of electrophysiological, biochemical, and histopathological examinations were performed. The results showed that HgS induced a 60% caloric response abnormality (40% caloric hyperfunction and 20% hypofunction), whereas the abnormal responses appeared to be more severe (six out of six) in the cinnabar group. The Hg contents of whole blood and cerebellum were increased and correlated to their neurotoxic effects on the VOR system, indicating that both insoluble HgS and cinnabar could be absorbed from the gastrointestinal tract and distributed to the cerebellum. Although the vestibular labyrinth revealed no remarkable change under light microscopy, loss of Purkinje cells in the cerebellum was detected, and the enzymatic Na(+)/K(+)-ATPase activity of cerebellum (a higher inhibitory center of the VOR system) was significantly inhibited by HgS and cinnabar. Moreover, cerebellar nitric oxide (NO) production was increased significantly. Hence, we tentatively conclude that the increased Hg contents in the cerebellum following oral administration of HgS and cinnabar were responsible, at least in part, for the detrimental neurotoxic effect on the VOR system. Potentially, decreasing Na(+)/K(+)-ATPase activity and increasing NO production within the cerebellar regulatory center are postulated to mediate this VOR dysfunction caused by the mercurial compounds and cinnabar.

Temporal bone pathology in fetuses exposed to isotretinoin.

Isotretinoin can be teratogenic, affecting many tissues, including the ear. However, there are only two histopathologic studies of the temporal bone in affected humans, and neither describes the findings in early gestation. We had the opportunity to study both temporal bones in each of two fetuses (22 and 24 weeks) exposed to isotretinoin in early gestation. One of the fetuses had a dilated IVth ventricle and a hypoplastic cerebellar vermis, while no dysmorphic features were seen in the other. In both infants the external ears were not noticeably abnormal. Histologically, anomalies of the middle ear included medial deviation of the malleus, forward displacement of the incus, and a small tympanic cavity (4/4); unilateral absence of the stapes (1/4); single "columella" crus and hypoplastic footplate (3/4); and unilateral dehiscence of the facial canal in one infant. Autolysis limited the examination of the labyrinth, but there was reduction in the number of cochlear spirals, and dilatation of the saccule in both infants. Anomalies of the middle and inner ear can be present without anomalies of the external ear or the central nervous system, and may be found even after relatively short exposures. These anomalies are similar to those detected in experimental exposure to isotretinoin, and are consistent with altered expression of the goosecoid gene.