- Original Articles
- Open Access
Expression of Human GLI in Mice Results in Failure to Thrive, Early Death, and Patchy Hirschsprung-like Gastrointestinal Dilatation
© Picower Institute Press 1997
- Accepted: 20 October 1997
- Published: 1 December 1997
GLI is an oncodevelopmental gene in the vertebrate hedgehog/patched signaling pathway that is spatiotemporally regulated during development and is amplified in a subset of human cancers. GLI is the prototype for the Gli-Kruppel family of transcription factors, which includes the Drosophila segment polarity gene ci, the C. elegans sex-deteimining gene tra-1, and human and mouse GLI3, all of which contain a conserved domain of five C2-H2 zinc fingers. GLI3 mutations have been implicated in the mouse mutant extra toes, as well as in human Greig cephalopolydactaly syndrome and the autosomal dominant form of Pallister-Hall syndrome. As such, GLI and the vertebrate hedgehog/patched signaling pathway appear to play important roles in both normal development and neoplasia.
Materials and Methods
Since it is not known whether aberrant GLI expression is similarly linked to developmental disorders, we developed gain-of-function transgenic mice which express human GLI ectopically.
Affected transgenic mice exhibit a phenotype of failure to thrive, early death, and Hirschsprung-like patches of gastrointestinal dilatation. The colons of affected mice have greatly attenuated smooth muscle layers and abnormal overlying epithelium. The density of myenteric plexuses is reduced in the colonic walls. The severity of the phenotype is related to the level of transgene expression.
The transgenic mouse model supports a role for GLI in gastrointestinal development. As part of the vertebrate hedgehog/patched signaling pathway, GLI is essential to mesoderm and CNS ectoderm development and transgenic GLI expression affects neuronal, muscular, and epithelial cell differentiation in the gut. Expression of human GLI in mice results in impairment of enteric neuronal development and a Hirschsprung-like phenotype.
GLI is the prototypical gene of the Gli-Kruppel family of transcription factors and is a mediator of hedgehog/patched signaling (1–4). The pathway is highly conserved, with homologous genes present in C. elegans, Drosophila, chick, mouse, and human (5–7). The vertebrate hedgehog/patched signaling pathway has been shown to be essential for normal development (8,9). Mutations of the genes Sonic Hedgehog, Patched, and GLI3 cause the human disorders holoprosencephaly (10), basal cell nevus syndrome (1), and Greig cephalopolydactyly syndrome (12) and Pallister-Hall syndrome (13–15), respectively. In vertebrates there are three GLI genes based on sequence homology in the region coding for the zinc finger DNA binding domains; GLI, GLI2, and GLI3. The human GLI gene has not yet been linked to a known developmental disorder, but its amplification in some gliomas, osetosarcomas, and rhabdomyosarcomas suggests an important role in growth regulation (16).
The human GLI gene is expressed in the human embryonal carcinoma cell lines Tera-1 and NTera-2 but not in normal adult tissues, except for very low levels in testes, myometrium, cerebellum, and oviduct (3,17). To localize the expression of mouse gli during development, in situ hybridization studies were performed on wild-type mouse embryos. In E7.5 mouse embryos, gli transcripts were present in ectoderm and mesoderm, but not in endoderm. In E10 through E18 embryos, gli transcripts were present in the primordia of the central nervous system, skeletal system, and the gastrointestinal (GI) tract. Expression was evident in the mesoderm of developing GI tract, Meckel’s precartilage mesenchyme, the basis occipitus, rib mesenchymal condensations, primordial vertebral bodies, digital mesenchymal condensations in forefoot and hindfoot plates, the ependymal layer of the spinal cord, most anterior and ventral parts of the telencephalon, several regions of the ventral mesencephalon, and the developing choroid plexus (8,9).
Here we report the production of transgenic mice which express human GLI. The transgenic animals display a complex phenotype related to the level of transgene expression, which includes abnormal development of the colon and patchy Hirschsprung-like dilatation. The colonic defect is associated with abnormal epithelium and decreased density of myenteric plexuses.
To generate gain-of-function mice, full-length human GLI cDNA was ligated to the inducible 770 bp mouse metallothionein-1 (pMT-1) promoter. An SV40 small t intron was inserted between pMT-1 and GLI, and the plasmid was cut with BssHII to yield a 4.7 kb fusion gene for microinjection. We then microinjected 2 µg/µl DNA into the pronucleus of one-cell ICR mouse embryos, which were transferred to pseudopregnant CB6F1 surrogate mothers. Southern blot analysis of mouse tail DNA identified one male founder, which was bred to a wild-type ICR female. The GLI transgene showed Mendelian transmission, and hemizygous Fl siblings (GLI/+) were mated to produce homozygous transgenic mice (GLI/GLI), which were identified by Southern blot analysis. Homozygosity was confirmed by analysis of the test-cross offspring.
One-half centimeter of mouse tail was incubated in 1 ml lysis buffer (50 mM Tris, pH 9.0, 50 mM EDTA, 0.4 M NaCl, 5 mM DTT, 2.5 mM Spermidine, 1% SDS, 1 mg/ml fresh Proteinase K) at 55°C overnight. The supernatant was mixed with 5 ml lysis buffer B (50 mM Tris, pH 8.0, 50 mM EDTA, 400 mM NaCl) and 2 ml saturated NaCl. After centrifuging, 5 ml isopropanol was added to the supernatant and mixed by swirling. The DNA was redissolved in TE.
Total RNA or poly (A)+ RNA were isolated with Qiagen RNeasy™ or Oligotex™ mRNA kits, respectively. For reverse transcription-polymerase chain reaction (RT-PCR), total RNA was treated with RNase-free DNase, which was removed from the RNA samples with RNeasy™ columns.
Southern Hybridization Analysis
Five micrograms of genomic DNA from each mouse was digested with BamHI and XbaI and electrophoresed on 0.8% agarose gels, then transferred to Bio-Rad Zeta-probe membranes in 0.4 M NaOH for 2–3 hr. The membranes were air-dried and baked at 80°C for 30 min. The probe for Southern analysis was a 2.1 kb fragment of human GLI cDNA cut with XhoII and was labeled with a random primer labeling kit at 109 cpm/µg specific activity. The membranes were prewet with hybridization solution (0.5 M NaH2PO4, pH 7.2 and 7% SDS) and hybridized at 65°C overnight in 3 ml of hybridization solution; the final probe concentration was 2 × 106 cpm/ml. After hybridization, the membranes were washed three times at 65°C in 20 mM Na2HPO4, pH 7.2, 1% SDS solution.
One hundred nanograms of total RNA or 50 ng of poly (A)+ RNA was treated with RNase-free DNase I (Boehringer Mannheim), and RT-PCR was performed using the GeneAmp RNA PCR kit (Perkin Elmer Cetus). The reverse transcription reaction was performed at room temperature for 10 min for annealing and at 42°C for 50 min for synthesis of cDNA, then at 99°C for 5 min, and finally placed on ice until the reaction buffer for PCR amplification was added. Primers and polymerase were added for PCR amplification. The method distinguished between endogenous and transgene expression by using human-specific primers (5′-GACCATGCACTGTCTTGACA-3′ and 5′-AGTCATACTCACGCCTCGAA-3′) to amplify a 238 bp fragment of the transgene.
RT-PCR reactions were carried out using five concentrations of RNA. Band intensities were quantified using NIH Image after digital scanning. Linear response ranges were established for both actin and the transgene RT-PCR product. Actin-to-transgene band intensity ratios were established and compared using data points from the 10-ng reactions, which were within the linear ranges of both actin and the transgene.
Generation of GLI Gain-of-Function Transgenic Mice
GLI Transgenic Mice Exhibit Failure to Thrive, Premature Death, and Hirschsprung-like Gastrointestinal Dilatation
Number of myenteric plexuses/mm in transverse sections of colon
Healthy Tg with Zn
Sick Tg with Zn
Sick Tg; normal wall
Sick Tg; transitional wall
Sick Tg; thin wall
Number of myenteric plexuses
5.1 ± 1.1
1.9 ± 1.3
1.2 ± 1.4
4.3 ± 1.2
3.1 ± 1.0
0.3 ± 0.6
Other tissues known to express GLI in normal development were not shown to be affected in the transgenic mice. A complete histologic evaluation of the central nervous system of a 3-month-old transgenic animal showed no abnormalities, and X-ray examination showed no changes in the skeleton. Blood panels and chemistries were all within normal limits, with no evidence of renal or hepatic failure that might have accounted for the failure-to-thrive phenotype. On necropsy, there was no evidence of bowel perforation, peritonitis, or gross mechanical obstruction.
Phenotype Severity Correlates with Increased GLI Transgene Expression
Quantitative RT-PCR studies showed that zinc induction was clearly linked to transgene expression and that the severity of the phenotype was related to the level of transgene GLI expression (Fig. 4C, D). Homozygous zinc-induced transgenic mice demonstrating the “sick” phenotype (small size, poor grooming, poor mobility) showed a 3.1-fold increase in intestinal transgene GLI RNA levels compared with healthy, noninduced mice. Healthy transgenic animals induced with zinc expressed 2.5 times the amount of intestinal transgene RNA compared with their noninduced healthy transgenic counterparts. Liver tissue from noninduced animals also showed that the quantity of transgene RNA correlated with phenotype; sick animals demonstrated 5.1 times the transgene RNA of healthy ones. In adult mice, endogenous mouse gli expression is limited to the brain, testes, and uterus. These results show that message levels have an effect on the phenotype of the transgenic animals.
These results demonstrate that expression of human GLI cDNA in mice can lead to a distinctive phenotype involving failure to thrive, premature death, and patchy Hirschsprung-like gastrointestinal dilatation. In this model, dysregulation of GLI has a profound effect. There is dysmorphogenesis of the colon of the animals with decreased density of myenteric plexuses in the colonic wall. The gain-of-function phenotype is transgene dose-dependent, as demonstrated by RT-PCR of transgene expression levels. The phenotype of the zinc-induced homozygous mice is more extreme than that of the noninduced animals.
Sonic hedgehog (Shh), one of the vertebrate homologs of Drosophila hedgehog (hh), is the ligand for the patched (ptc) receptor. In flies, hh regulates a negative feedback loop of ptc with ci in which ci activates the expression of dpp and ptc (23). GLI is the vertebrate homolog of ci, and Bmp-2/4 are the vertebrate homologs of dpp. Dysregulation of dpp in Drosophila disrupts normal midgut constriction (21,24), and dysregulation of Shh causes ectopic expression of Bmp-4 and Hoxd genes in chick hindgut mesoderm (25). Mouse gli is highly expressed in the mesodermal layer of normal GI tract from E13 until birth (8,26). Thus, the essential elements of a mesoderm-epithelial signaling network in the GI tract involving Shh, ptc, gli, and Bmp-4 are conserved from Drosophila to vertebrates. Therefore, ectopic expression of GLI would be expected to disrupt the pathway and result in abnormal gut development; this is precisely the result obtained in the present study. A similar but more posterior phenotype to the one presented here is obtained when Hoxd13 is knocked out in mice (27).
In humans, disruption of Shh results in holoprosencephaly, a very serious dysmorphogenic syndrome with numerous midline defects and axial skeletal abnormalities (10). These patterning defects, including cyclopia, are recapitulated in mice with null mutations of Shh (28). Deletions in the 3′ end of GLI3 in humans results in Pallister-Hall syndrome, manifesting hypothalamic hamartoma, hypopituitarism, polydactyly, imperforate anus, and renal and lung anomalies (13). There is a variant of Pallister Hall syndrome which includes holoprosencephaly and Hirschsprung’s disease (14,15); the genetic defect responsible for this variation is not known, but our results indicate that GLI is a candidate gene.
The role of GLI in GI tract development remains to be established, but since GLI has been placed in the vertebrate hedgehog/patched pathway (11,29), it is reasonable to suppose that GLI is part of a signaling cascade that is important in mesoderm development and in turn affects neuronal, muscular, and epithelial cell differentiation in the gut. In this model, it is likely that impairment of enteric neuronal development led to the Hirschsprung-like phenotype.
We thank Dr. M. DalCanto for performing the CNS histologic evaluation of the transgenic mice and Dr. P. Chou for histologic evaluation of GI tract walls. This work was aided by grants 93-32 and 94-51 from the American Cancer Society, Illinois Division, Inc. and was supported in part by Public Health Service grant CA64395 from the National Cancer Institute and HD28992 from the National Institute of Child Health and Human Development.
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