Robert J. Lipinski

Dose- and Route-Dependent Teratogenicity, Toxicity, and Pharmacokinetic Profiles of the Hedgehog Signaling Antagonist Cyclopamine in the Mouse

The Hedgehog (Hh) signaling pathway is an essential regulator of embryonic development and appears to play important roles in postnatal repair and cancer progression and metastasis. The teratogenic Veratrum alkaloid cyclopamine is a potent Hh antagonist and is used experimentally both in vitro and in vivo to investigate the role of Hh signaling in diverse biological processes. Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. The dysmorphogenic concentration of cyclopamine was determined in vitro via mouse whole-embryo culture assays to be 2.0 microM. We administered cyclopamine to female C57BL/6J mice at varied doses by oral gavage, ip injection, or osmotic pump infusion and assessed toxicity and pharmacokinetic (PK) models. Bolus administration was limited by toxicity and rapid clearance. In vivo cyclopamine infusion at 160 mg/kg/day yielded a dam serum steady-state concentration of approximately 2 microM with a corresponding amniotic fluid concentration of approximately 1.5 microM. Gross facial defects were induced in 30% of cyclopamine-exposed litters, with affected embryos exhibiting cleft lip and palate. This is the first report describing the PKs and teratogenic potential of cyclopamine in the mouse and demonstrates that transient Hh signaling inhibition induces facial clefting anomalies in the mouse that mimic common human birth defects.

Ethanol-induced face-brain dysmorphology patterns are correlative and exposure-stage dependent.

Prenatal ethanol exposure is the leading preventable cause of congenital mental disability. Whereas a diagnosis of fetal alcohol syndrome (FAS) requires identification of a specific pattern of craniofacial dysmorphology, most individuals with behavioral and neurological sequelae of heavy prenatal ethanol exposure do not exhibit these defining facial characteristics. Here, a novel integration of MRI and dense surface modeling-based shape analysis was applied to characterize concurrent face-brain phenotypes in C57Bl/6J fetuses exposed to ethanol on gestational day (GD)7 or GD8.5. The facial phenotype resulting from ethanol exposure depended upon stage of insult and was predictive of unique patterns of corresponding brain abnormalities. Ethanol exposure on GD7 produced a constellation of dysmorphic facial features characteristic of human FAS, including severe midfacial hypoplasia, shortening of the palpebral fissures, an elongated upper lip, and deficient philtrum. In contrast, ethanol exposure on GD8.5 caused mild midfacial hypoplasia and palpebral fissure shortening, a shortened upper lip, and a preserved philtrum. These distinct, stage-specific facial phenotypes were associated with unique volumetric and shape abnormalities of the septal region, pituitary, and olfactory bulbs. By demonstrating that early prenatal ethanol exposure can cause more than one temporally-specific pattern of defects, these findings illustrate the need for an expansion of current diagnostic criteria to better capture the full range of facial and brain dysmorphology in fetal alcohol spectrum disorders.

Magnetic Resonance-based imaging in animal models of Fetal Alcohol Spectrum Disorder

Magnetic resonance imaging (MRI) techniques, such as magnetic resonance microscopy (MRM), diffusion tensor imaging (DTI), and magnetic resonance spectroscopy (MRS), have recently been applied to the study of both normal and abnormal structure and neurochemistry in small animals. Herein, findings from studies in which these methods have been used for the examination of animal models of Fetal Alcohol Spectrum Disorder (FASD) are discussed. Emphasis is placed on results of imaging studies in fetal and postnatal mice that have highlighted the developmental stage dependency of prenatal ethanol exposure-induced CNS defects. Consideration is also given to the promise of methodological advances to allow in vivo studies of aberrant brain and behavior relationships in model animals and to the translational nature of this work.

The teratogenic effects of prenatal ethanol exposure are exacerbated by Sonic Hedgehog or GLI2 haploinsufficiency in the mouse.

Disruption of the Hedgehog signaling pathway has been implicated as an important molecular mechanism in the pathogenesis of fetal alcohol syndrome. In severe cases, the abnormalities of the face and brain that result from prenatal ethanol exposure fall within the spectrum of holoprosencephaly. Single allele mutations in the Hh pathway genes Sonic Hedgehog (SHH) and GLI2 cause holoprosencephaly with extremely variable phenotypic penetrance in humans. Here, we tested whether mutations in these genes alter the frequency or severity of ethanol-induced dysmorphology in a mouse model. Timed pregnancies were established by mating Shh+/− or Gli2+/− male mice backcrossed to C57BL/6J strain, with wildtype females. On gestational day 7, dams were treated with two ip doses of 2.9 g/kg ethanol (or vehicle alone), administered four hrs apart. Fetuses were then genotyped and imaged, and the severity of facial dysmorphology was assessed. Following ethanol exposure, mean dysmorphology scores were increased by 3.2- and 6.6-fold in Shh+/− and Gli2+/− groups, respectively, relative to their wildtype littermates. Importantly, a cohort of heterozygous fetuses exhibited phenotypes not typically produced in this model but associated with severe holoprosencephaly, including exencephaly, median cleft lip, otocephaly, and proboscis. As expected, a correlation between the severity of facial dysmorphology and medial forebrain deficiency was observed in affected animals. While Shh+/− and Gli2+/− mice have been described as phenotypically normal, these results illustrate a functional haploinsufficiency of both genes in combination with ethanol exposure. By demonstrating an interaction between specific genetic and environmental risk factors, this study provides important insights into the multifactorial etiology and complex pathogenesis of fetal alcohol syndrome and holoprosencephaly.

Cleft lip and palate results from Hedgehog signaling antagonism in the mouse: Phenotypic characterization and clinical implications

BACKGROUND The Hedgehog (Hh) pathway provides inductive signals critical for developmental patterning of the brain and face. In humans and in animal models interference with this pathway yields birth defects, among the most well-studied of which fall within the holoprosencephaly (HPE) spectrum. METHODS Timed-pregnant C57Bl/6J mice were treated with the natural Hh signaling antagonist cyclopamine by subcutaneous infusion from gestational day (GD) 8.25 to 9.5, or with a potent cyclopamine analog, AZ75, administered by oral gavage at GD 8.5. Subsequent embryonic morphogenesis and fetal central nervous system (CNS) phenotype were respectively investigated by scanning electron microscopy and high resolution magnetic resonance imaging (MRI). RESULTS In utero Hh signaling antagonist exposure induced a spectrum of craniofacial and brain malformations. Cyclopamine exposure caused lateral cleft lip and palate (CLP) defects attributable to embryonic deficiency of midline and lower medial nasal prominence tissue. The CLP phenotype was accompanied by olfactory bulb hypoplasia and anterior pituitary aplasia, but otherwise grossly normal brain morphology. AZ75 exposure caused alobar and semilobar HPE with associated median facial deficiencies. An intermediate phenotype of median CLP was produced infrequently by both drug administration regimens. CONCLUSIONS The results of this study suggest that interference with Hh signaling should be considered in the CLP differential and highlight the occurrence of CNS defects that are expected to be present in a cohort of patients having CLP. This work also illustrates the utility of fetal MRI-based analyses and establishes a novel mouse model for teratogen-induced CLP.

Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

BackgroundHedgehog (Hh) signaling is a conserved morphogenetic pathway which plays critical roles in embryonic development, with emerging evidence also supporting a role in healing and repair processes and tumorigenesis. The Gli family of transcription factors (Gli1, 2 and 3) mediate the Hedgehog morphogenetic signal by regulating the expression of downstream target genes. We previously characterized the individual and cooperative roles of the Gli proteins in Hh target gene regulation using a battery of primary embryonic fibroblasts from Gli null mice.ResultsHere, we describe the establishment of spontaneously immortalized mouse embryonic fibroblast (iMEF) cell lines lacking single and multiple Gli genes. These non-clonal cell lines recapitulate the unique ligand mediated transcriptional response of primary MEFs. While loss of Gli1 had no effect on target gene induction, Gli2 null cells demonstrated reduced target gene induction while Gli3 null cells exhibited elevated basal and ligand-induced expression. Target gene response in Gli1-/-2-/- iMEFs was severely reduced while Gli2-/-3-/- iMEFs were incapable of ligand-induced transcriptional response. However, we found that both Gli1-/-2-/- and Gli2-/-3-/- iMEFs exhibited robust leukotriene synthesis-dependent migration responses to Hh ligand, demonstrating that this response is not transcriptionally-dependent.ConclusionThis study provides fundamental characterizations of the transcriptional and non-transcriptional Hh responsiveness of a battery of Gli-null iMEFs. Moving forward, these cell lines should prove a valuable tool set to study the unique functional regulation of the Gli proteins in a Hh-responsive cell-type.

Genesis of teratogen‐induced holoprosencephaly in mice

Evidence from mechanical, teratological, and genetic experimentation demonstrates that holoprosencephaly (HPE) typically results from insult prior to the time that neural tube closure is completed and occurs as a consequence of direct or indirect insult to the rostral prechordal cells that induce the forebrain or insult to the median forebrain tissue, itself. Here, we provide an overview of normal embryonic morphogenesis during the critical window for HPE induction, focusing on the morphology and positional relationship of the developing brain and subjacent prechordal plate and prechordal mesoderm cell populations. Subsequent morphogenesis of the HPE spectrum is then examined in selected teratogenesis mouse models. The temporal profile of Sonic Hedgehog expression in rostral embryonic cell populations and evidence for direct or indirect perturbation of the Hedgehog pathway by teratogenic agents in the genesis of HPE is highlighted. Emerging opportunities based on recent insights and new techniques to further characterize the mechanisms and pathogenesis of HPE are discussed.

Sonic hedgehog signaling regulates the expression of insulin-like growth factor binding protein-6 during fetal prostate development.

At the onset of ductal morphogenesis in the developing prostate, Shh expression condenses at evaginations of urogenital sinus epithelium and activates Gli transcription factors in the adjacent mesenchyme. Abrogation of Hedgehog signaling disrupts proper prostatic budding, ductal growth, and branching. We now show that Hedgehog signaling regulates the expression of insulin‐like growth factor binding protein‐6 (Igfbp‐6) in the developing mouse prostate. Igfbp‐6 is a secreted factor that specifically binds insulin‐like growth factor‐II (IGF‐II), prevents its binding to the IGF‐I receptor, and is thought to regulate the activity of IGF‐II in growth and differentiation. Igfbp‐6 is expressed in both the developing and adult prostate. In the urogenital sinus, Igfbp‐6 mRNA colocalized with Ptc1 and Gli1 mRNA in the mesenchyme, while Igfbp‐6 protein was found in both the mesenchymal and epithelial layers. Exogenous Shh peptide induced expression of Igfbp‐6 in the developing prostate while the chemical inhibitor of Hedgehog signaling, cyclopamine, reduced its expression. These studies show that Igfbp‐6 is an actual target of Shh signaling in the urogenital sinus and provide the first evidence for a linkage between the Hedgehog and IGF signaling pathways in prostate development. Developmental Dynamics 233:829–836, 2005.

Definition of Critical Periods for Hedgehog Pathway Antagonist-Induced Holoprosencephaly, Cleft Lip, and Cleft Palate

The Hedgehog (Hh) signaling pathway mediates multiple spatiotemporally-specific aspects of brain and face development. Genetic and chemical disruptions of the pathway are known to result in an array of structural malformations, including holoprosencephaly (HPE), clefts of the lip with or without cleft palate (CL/P), and clefts of the secondary palate only (CPO). Here, we examined patterns of dysmorphology caused by acute, stage-specific Hh signaling inhibition. Timed-pregnant wildtype C57BL/6J mice were administered a single dose of the potent pathway antagonist vismodegib at discrete time points between gestational day (GD) 7.0 and 10.0, an interval approximately corresponding to the 15th to 24th days of human gestation. The resultant pattern of facial and brain dysmorphology was dependent upon stage of exposure. Insult between GD7.0 and GD8.25 resulted in HPE, with peak incidence following exposure at GD7.5. Unilateral clefts of the lip extending into the primary palate were also observed, with peak incidence following exposure at GD8.875. Insult between GD9.0 and GD10.0 resulted in CPO and forelimb abnormalities. We have previously demonstrated that Hh antagonist-induced cleft lip results from deficiency of the medial nasal process and show here that CPO is associated with reduced growth of the maxillary-derived palatal shelves. By defining the critical periods for the induction of HPE, CL/P, and CPO with fine temporal resolution, these results provide a mechanism by which Hh pathway disruption can result in “non-syndromic” orofacial clefting, or HPE with or without co-occurring clefts. This study also establishes a novel and tractable mouse model of human craniofacial malformations using a single dose of a commercially available and pathway-specific drug.

Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells

BackgroundHedgehog (Hh) signaling from the urogenital sinus (UGS) epithelium to the surrounding mesenchyme plays a critical role in regulating ductal formation and growth during prostate development. The primary cilium, a feature of most interphase vertebrate cell types, serves as a required localization domain for Hh signaling transducing proteins.ResultsImmunostaining revealed the presence of primary cilia in mesenchymal cells of the developing prostate. Cell-based assays of a urongenital sinus mesenchymal cell line (UGSM-2) revealed that proliferation-limiting (serum starvation and/or confluence) growth conditions promoted cilia formation and correlated with pathway activation associated with accumulation of Smoothened in primary cilia. The prostate cancer cell lines PC-3, LNCaP, and 22RV1, previously shown to lack demonstrable autocrine Hh signaling capacity, did not exhibit primary cilia even under proliferation-limiting growth conditions.ConclusionWe conclude that paracrine Hedgehog signaling activity in the prostate is associated with the presence of primary cilia on stromal cells but that a role in autocrine Hh signaling remains speculative.