Iduronate-2-sulfatase transport vehicle rescues behavioral and skeletal phenotypes in a mouse model of Hunter syndrome.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in cellular accumulation of glycosaminoglycans (GAGs) throughout the body. Treatment of MPS II remains a considerable challenge as current enzyme replacement therapies do not adequately control many aspects of the disease, including skeletal and neurological manifestations. We developed an IDS transport vehicle (ETV:IDS) that is engineered to bind to the transferrin receptor; this design facilitates receptor-mediated transcytosis of IDS across the blood-brain barrier and improves its distribution into the brain while maintaining distribution to peripheral tissues. Here we show that chronic systemic administration of ETV:IDS in a mouse model of MPS II reduced levels of peripheral and central nervous system GAGs, microgliosis, and neurofilament light chain, a biomarker of neuronal injury. Additionally, ETV:IDS rescued auricular and skeletal abnormalities when introduced in adult MPS II mice. These effects were accompanied by improvements in several neurobehavioral domains, including motor skills, sensorimotor gating, and learning and memory. Together, these results highlight the therapeutic potential of ETV:IDS for treating peripheral and central abnormalities in MPS II. DNL310, an investigational ETV:IDS molecule, is currently in clinical trials as a potential treatment for patients with MPS II.

Agaricus bisporus-Derived Glucosamine Hydrochloride Facilitates Skeletal Injury Repair through Bmp Signaling in Zebrafish Osteoporosis Model.

Glucosamine hydrochloride (GAH), one of the most basic and important derivatives of chitin, is obtained by hydrolysis of chitin in concentrated hydrochloric acid. At present, little is known about how GAH functions in skeletal development. In this report, we demonstrate that GAH, extracted from the cell wall of Agaricus bisporus, acts in a dose-dependent manner to promote not only cartilage and bone development in larvae but also caudal fin regeneration in adult fish. Furthermore, GAH treatment causes a significant increase in expression of bone-related marker genes, indicating its important role in promoting skeletal development. We show that in both larval and adult osteoporosis models induced by high iron osteogenic defects are significantly ameliorated after treatment with GAH, which regulates expression of a series of bone-related genes. Finally, we demonstrate that GAH promotes skeletal development and injury repair through bone morphogenetic protein (Bmp) signaling, and it works at the downstream of the receptor level. Taken together, our findings not only provide a strong research foundation and strategy for the screening of natural osteoporosis drugs and product development using a zebrafish model but also establish the potential for the development of Agaricus bisporus-derived GAH as a new drug for osteoporosis treatment.

Canonical and noncanonical TGF-ß signaling regulate fibrous tissue differentiation in the axial skeleton.

Previously, we showed that embryonic deletion of TGF-ß type 2 receptor in mouse sclerotome resulted in defects in fibrous connective tissues in the spine. Here we investigated how TGF-ß regulates expression of fibrous markers: Scleraxis, Fibromodulin and Adamtsl2. We showed that TGF-ß stimulated expression of Scleraxis mRNA by 2 h and Fibromodulin and Adamtsl2 mRNAs by 8 h of treatment. Regulation of Scleraxis by TGF-ß did not require new protein synthesis; however, protein synthesis was required for expression of Fibromodulin and Adamtsl2 indicating the necessity of an intermediate. We subsequently showed Scleraxis was a potential intermediate for TGF-ß-regulated expression of Fibromodulin and Adamtsl2. The canonical effector Smad3 was not necessary for TGF-ß-mediated regulation of Scleraxis. Smad3 was necessary for regulation of Fibromodulin and Adamtsl2, but not sufficient to super-induce expression with TGF-ß treatment. Next, the role of several noncanonical TGF-ß pathways were tested. We found that ERK1/2 was activated by TGF-ß and required to regulate expression of Scleraxis, Fibromodulin, and Adamtsl2. Based on these results, we propose a model in which TGF-ß regulates Scleraxis via ERK1/2 and then Scleraxis and Smad3 cooperate to regulate Fibromodulin and Adamtsl2. These results define a novel signaling mechanism for TGFß-mediated fibrous differentiation in sclerotome.

OPG-Fc treatment partially rescues low bone mass phenotype in mature Bgn/Fmod deficient mice but is deleterious to the young mouse skeleton.

Biglycan (Bgn) and Fibromodulin (Fmod) are small leucine rich proteoglycans (SLRPs) which are abundant in the extra-cellular matrix (ECM) of mineralized tissues. We have previously generated a Bgn/Fmod double knock-out (DKO) mouse model and found it has a 3-fold increase in osteoclastogenesis compared with Wild type (WT) controls, resulting in a markedly low bone mass (LBM) phenotype. To try and rescue/repair the LBM phenotype of Bgn/Fmod DKO mice by suppressing osteoclast formation and activity, 3- and 26-week-old Bgn/Fmod DKO mice and age/gender matched WT controls were treated with OPG-Fc for 6 weeks after which bone parameters were evaluated using DEXA, micro-computed tomography (µCT) and serum biomarkers analyses. In the appendicular skeleton, OPG-Fc treatment improved some morphometric and geometric parameters in both the trabecular and cortical compartments in Bgn/Fmod DKO female and male mice, especially in the repair module. For many of the skeletal parameters analyzed, the Bgn/Fmod DKO mice were more responsive to the treatment than their WT controls. In addition, we found that OPG-Fc treatment was not able to prevent or ameliorate the formation of ectopic ossification, which are common lesions seen in aged joints and are one of the phenotypical hallmarks of our Bgn/Fmod DKO model. Analysis of skull bones, specifically the occipital bone, showed the treatment recovered some parameters of LBM phenotype in the craniofacial skeleton, more so in the younger rescue module. Using OPG-Fc as treatment alleviated, yet did not completely restore, the severe osteopenia and mineralized tissue structural abnormalities that Bgn/Fmod DKO mice suffer from.

Impact of feeding microalgae (Aurantiochytrium limacinum) and co-extruded mixture of full-fat flaxseed as sources of n-3 fatty acids to ISA brown and Shaver white breeders and progeny on pullet skeletal attributes at hatch through to 18 weeks of age.

Impact of feeding n-3 fatty acids (FA) to ISA brown and Shaver white breeders and their progeny on bone development in pullets was investigated. Breeders were fed Control (CON); CON + 1% microalgae (DMA: Aurantiochytrium limacinum) as the source of docosahexaenoic acid; and CON + 2.6% of a co-extruded mixture of full-fat flaxseed (FFF) and pulses mixture as source of α-linolenic acid. Test diets (DMA and FFF) were balanced for total n-3 FA and n-6: n-3 FA ratio. Samples of day-old progeny were euthanized for bone mineral content (BMC) and tibia collagen type II. The remaining pullets were fed posthatch treatments as follows: from breeder CON: CON (CON-CON), DMA (CON-DMA), and FFF (CON-FFF), from breeder DMA: CON (DMA-CON) and DMA (DMA-DMA) and from breeder FFF: CON (FFF-CON) and FFF (FFF-FFF). A total of 60 pullets per posthatch diets were reared in cages (12 pullets/cage, n = 5) with free access to feed and water, bled at 6, 12, and 18 wk of age (WOA) for bone turnover markers and necropsied at 18 WOA for tibia and femur samples. Day-old pullets from breeder fed CON had greater BMC (P < 0.001) relative to those from breeders fed other diets. There was strain and diet interaction (P ≤ 0.024) on tibia breaking strength (TBS) and tibia cortical ash concentration at 18 WOA such that diet responses were only observed in Shaver white pullets. In this context, TBS of DMA-DMA and FFF-FFF was greater than for pullets originating from CON breeder, and the cortical ash weight of DMA-DMA and FFF-FFF pullets was 23.8 and 20.2%, respectively, higher than for CON-CON pullets. In conclusions, the strain effects were strong on tibia attributes on 18-week-old pullets. Breeder feeding of n-3 FA was more effective when concomitant with posthatch feeding of n-3 FA in supporting the skeletal strength and cortical bone development in Shaver white pullets. Further investigations are warranted to establish the impact these strategies on skeletal health during laying cycle.

Effects of phthalate acid esters on zebrafish larvae: Development and skeletal morphogenesis.

This study evaluated the acute developmental toxicity of six priority phthalic acid esters (PAEs) including dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), di-2-ethylhexyl phthalate (DEHP), di-n-octyl phthalate (DNOP), and benzyl butyl phthalate (BBP) in zebrafish embryos. A novel alcian blue and alizarin red double staining was performed to detect skeletal development of zebrafish larvae. Results revealed that all six PAEs could induce different developmental abnormalities in zebrafish larvae, including abnormal movement, decreased heart rate, spinal curvature, and pericardial edema. The bone development of zebrafish larvae exposed to PAEs was also affected by PAEs acute exposure. Among PAEs, DBP, and BBP even at low doses can cause mortality in zebrafish, implying their higher toxicity. Contrarily, DEHP and DNOP showed minor effects on the developmental morphology of zebrafish larvae. However, the gene expression levels of skeleton-related genes showed the upregulation of the runx2b and shha genes after DEHP and DBP exposure. Taken together, the strict use and release of PAEs in the environment should be supervised by the government for ecological and environmental safety.

Long-Term Accumulation of Metals in the Skeleton as Related to Osteoporotic Derangements.

The concentrations of metals in the environment are still not within the recommended limits as set by the regulatory authorities in various countries because of human activities. They can enter the food chain and bioaccumulate in soft and hard tissues/organs, often with a long half-life of the metal in the body. Metal exposure has a negative impact on bone health and may result in osteoporosis and increased fracture risk depending on concentration and duration of metal exposure and metal species. Bones are a long-term repository for lead and some other metals, and may approximately contain 90% of the total body burden in birds and mammals. The present review focuses on the most common metals found in contaminated areas (mercury, cadmium, lead, nickel, chromium, iron, and aluminum) and their effects on bone tissue, considering the possibility of the long-term bone accumulation, and also some differences that might exist between different age groups in the whole population.

Ambroxol improves skeletal and hematological manifestations on a child with Gaucher disease.

Gaucher disease (GD) is a lysosomal storage disease caused by the deficiency of glucocerebrosidase characterized by a broad spectrum of clinical manifestations including hepatosplenomegaly, bone infiltration, and cytopenia, and even central nervous system involvement. Bone manifestations are typical of the GD-I and partially responded to mainstay therapy. Ambroxol (ABX), an approved cough-suppressant, was identified as an enzyme-enhancement agent of the residual activity of glucocerebrosidase mutants derived from different misfolding-mutations in the GBA gene. Here, we describe the early beneficial effects of ABX on skeletal and hematological manifestations of a child suffering with progressive GD-I.

Microdiet Formulation with Phospholipid Modulate Zebrafish Skeletal Development and Reproduction.

Dietary phospholipids' (PLs) content, origin, and profile are known to affect fish development and reproductive performance, but their effects in zebrafish (Danio rerio) nutrition are still poorly investigated. Therefore, this study aimed to assess the effect of practical microdiets containing plant-based and marine PL sources in zebrafish growth, survival, skeletal development, and reproductive performance. Reproductive performance was evaluated according to sperm motility, number of eggs, egg morphometry, hatching rate, and offspring standard length at 5 days postfertilization (dpf). For this purpose, seven microdiets were used, where two control diets were tested along with a supplementation with soybean lecithin (SL) as a plant-based PL source, and krill oil (KO) and copepod oil (CO) as marine PL sources, or in combinations (SLCO and SLKO). KO supplementation decreased larval growth performance and induced severe skeletal anomalies. SL supplementation reduced sperm total motility but improved offspring length at 5 dpf. CO supplementation increased sperm motility and the number of spawned eggs. Our results showed that a careful selection of the origin of dietary PL sources for microdiet formulation is critical to ensure adequate skeletal development and reproductive success. This study contributes to the improvement of zebrafish microdiet formulation and optimization of zebrafish husbandry practices.

High Affinity to Skeleton Rare Earth Doped Nanoparticles for Near-Infrared II Imaging.

As a newly noninvasive emerging modality, NIR-II fluorescence imaging (1000-1700 nm) has many advantages over conventional visible and NIR-I imaging (700-900 nm). Unfortunately, only a few NIR-II fluorophores are suitable for bone imaging. Here, we report an NIR-II fluorophore based on DSPE-mPEG encapsulated rare earth doped nanoparticles (RENPs@DSPE-mPEG), which shows inherent affinity to bone without linking any targeting ligands, and thus, it provides an alternative noninvasive and nonradiation strategy for skeletal system mapping and bone disease diagnoses. Interestingly, within the NIR-II window, imaging at a longer wavelength (1345 nm) provides a higher resolution and signal-to-noise ratio than imaging at 1064 nm, even though the quantum yield at 1064 nm is 2-fold higher than that at 1345 nm. Besides bone imaging, RENPs@DSPE-mPEG show an imaging application in blood vessels and lymph nodes. Importantly, RENPs@DSPE-mPEG can be internalized by circulating white blood cells. This finding may open a window to increase efficient nanoparticle delivery in the fields such as immunotherapy and improve the diagnostic and therapeutic efficacy of cancer-targeted nanoparticles in clinical applications.

Wnt Antagonists in Hematopoietic and Immune Cell Fate: Implications for Osteoporosis Therapies.

PURPOSE OF REVIEW: We reviewed the current literature on the roles of the Wnt antagonists sclerostin (Sost) and sclerostin-containing domain protein 1 (Sostdc1) on bone homeostasis, the relationship of the hypoxia-inducible factor (Hif) and von Hippel-Lindau (Vhl) pathways on Sost expression, and how changes in bone induced by depletion of Sost, Sostdc1, and Vhl affect hematopoietic cells. RECENT FINDINGS: B cell development is adversely affected in Sost-knockout mice and is more severely affected in Vhl-knockout mice. Inflammation in the Sost-/- bone microenvironment could alter hematopoietic stem cell behavior. Sostdc1-/- mice display defects in natural killer cell development and cytotoxicity. Depletion of Sost and Sostdc1 have effects on immune cell function that warrant investigation in patients receiving Wnt antagonist-depleting therapies for treatment of bone diseases. Additional clinical applications for manipulation of Wnt antagonists include cancer immunotherapies, stem cell transplantation, and directed differentiation to immune lineages.

Isomeric flavonoid aglycones derived from Epimedii Folium exerted different intensities in anti-osteoporosis through OPG/RANKL protein targets.

Two Epimedium-derived isomeric flavonoids, CIT and IT, had the therapeutic effect in osteopenic rats. However, it is difficult to expound their activity differences in anti-osteoporosis. This paper contrasted their anti-osteoporosis activity from the perspective of their affinity to OPG/RANKL protein targets. Molecular docking indicated that both of CIT and IT could interact with the hydrophobic pockets of OPG/RANKL, while CIT was easier and more stable to combine with RANKL. On the contrary, compared with CIT, IT was more inclined to combine with OPG and stay away from combining with RANKL. Subsequently, whether the interaction between isomeric flavonoids and OPG/RANKL targets promoted or suppressed bone resorption was undefined and which was validated by zebrafish embryo and ovariectomized rats in this paper. Compared with IT, the staining area and cumulative optical density of zebrafish skeleton were significantly increased after the treatment of CIT (0.1 µM, p < 0.05). Furthermore, CIT mainly reflected a more significant role in upregulating OPG (p < 0.05), downregulating RANKL (p < 0.05), reducing serum AKP and TRACP level (p < 0.05), enhancing bone biomechanical properties (p < 0.05), increasing bone mineral density (p < 0.05) and improving trabecular bone microarchitecture (p < 0.05) in osteoporotic rats. In conclusion, the combination of isomeric flavonoids (CIT/IT) and OPG/RANKL targets attenuated the excitation effects of OPG or RANKL on RANKL. Because CIT was more firmly combined with RANKL than IT, CIT had stronger anti-osteoporosis effect by inhibiting bone resorption.

Bone development in laboratory mammals used in developmental toxicity studies.

Evaluation of the skeleton in laboratory animals is a standard component of developmental toxicology testing. Standard methods of performing the evaluation have been established, and modification of the evaluation using imaging technologies is under development. The embryology of the rodent, rabbit, and primate skeleton has been characterized in detail and summarized herein. The rich literature on variations and malformations in skeletal development that can occur in the offspring of normal animals and animals exposed to test articles in toxicology studies is reviewed. These perturbations of skeletal development include ossification delays, alterations in number, shape, and size of ossification centers, and alterations in numbers of ribs and vertebrae. Because the skeleton is undergoing developmental changes at the time fetuses are evaluated in most study designs, transient delays in development can produce apparent findings of abnormal skeletal structure. The determination of whether a finding represents a permanent change in embryo development with adverse consequences for the organism is important in study interpretation. Knowledge of embryological processes and schedules can assist in interpretation of skeletal findings.

Osteoimmunology: Effects of Standard Orthopaedic Interventions on Inflammatory Response and Early Fracture Healing.

Achieving fracture union is highly dependent on the initial inflammatory phase of fracture healing, which is influenced by both the local and systemic inflammatory environments. The rapidly emerging field of osteoimmunology involves the study of the interactions between the immune system and the skeletal system. Recent research has advanced the current state of knowledge regarding the effects of the surrounding soft-tissue injury, fracture hematoma, and the method of fracture fixation on the inflammatory phase of fracture healing. Acute systemic inflammation, as seen in patients with polytrauma, and chronic systemic inflammation, as seen in patients with diabetes or rheumatoid arthritis, affects the inflammatory phase of fracture healing. The use of NSAIDs can influence early fracture healing. Understanding the effects of standard orthopaedic interventions on the local and systemic inflammatory responses and early fracture healing is important for optimizing fracture union.

Influence of growth hormone therapy on selected dental and skeletal system parameters.

INTRODUCTION: Growth hormone deficiency (GHD) is one of the main indications for growth hormone therapy. One characteristic of this disease is bone age delay in relation to the chronological age. Pituitary dysfunction negatively affects the growth and development of the jaws and teeth of the child. The secretion of endocrine glands regulates growth, development, and gender differentiation. It also controls the growth of bones and teeth, regulates metabolism of calcium and phosphate, proteins, lipids and carbohydrates. The primary role in the endocrine system is played by the pituitary gland which is responsible for the production of somatotropin [1]. Dysfunction of the pituitary gland has a negative effect on the growth and development of long bones in the body, and may have an adverse effect on the development of maxilla, mandible and dentition of a child. There is some information in the literature that dental age is delayed in short stature children; the replacement of deciduous teeth by permanent teeth is also delayed, and newly erupted permanent teeth often require orthodontic treatment. Applying hormonal therapy positively affects the process of replacement of dentition [2, 3, 4, 5, 6]. OBJECTIVES: The aim of the study was to assess bone and dental age, as well as analyze the state of dentition in children diagnosed with GH deficiency treated with growth hormone, depending on the duration of treatment. MATERIAL AND METHODS: The study material consisted of 110 children (27 males, 83 females), hospitalized for somatotropin hypopituitarism in the Department of Paediatric Endocrinology and Diabetology at the Medical University of Lublin, Poland. The mean birth age was 13 years (156 months) with a standard deviation of 2 years and 6 months (30 months). 47 children (43%) started treatment with the growth hormone (group starting treatment) and 63 children (57%) whose treatment was started 2-3 years previously (group in the course of treatment). The control group consisted of 41 generally healthy children (15males, 25 females) with ENT problems, such as hypoacusis and a condition after nasal injury, hospitalized in the Department of Paediatric Otolaryngology at the Medical University of Lublin, Poland. The mean age was 11 years and 5 months (137 months) with standard deviation of 2 years and 5 months (29 months). Informed consent was obtained from the parents. The study was approved by the Bioethical Committee at the Medical University of Lublin (Resolution No. KE-0254 /216 /2012).

Maternal and developmental toxicity of the hallucinogenic plant-based beverage ayahuasca in rats.

Rats were treated orally with ayahuasca (AYA) on gestation days (GD) 6-20 at doses corresponding to one-(1X) to eight-fold (8X) the average dose taken by a human adult in a religious ritual, and the pregnancy outcome evaluated on GD21. Rats treated with 4X and 8X doses died during the treatment period (44 and 52%), and those that survived showed kidney injury. Rats surviving the 8X dose showed neuronal loss in hippocampal regions and in the raphe nuclei, and those from the 2X dose neuronal loss in CA1. Delayed intrauterine growth, induced embryo deaths and increased occurrence of foetal anomalies were observed at the 8X dose. At non-lethal doses, AYA enhanced embryolethality and the incidence of foetal soft-tissue and skeleton anomalies. This study suggested that AYA is developmentally toxic and that its daily use by pregnant women may pose risks for the conceptus.

Osteoporosis in Rheumatic Diseases: Anti-rheumatic Drugs and the Skeleton.

Osteoporosis in rheumatic diseases is a very well-known complication. Systemic inflammation results in both generalized and localized bone loss and erosions. Recently, increased knowledge of inflammatory process in rheumatic diseases has resulted in the development of potent inhibitors of the cytokines, the biologic DMARDs. These treatments reduce systemic inflammation and have some effect on the generalized and localized bone loss. Progression of bone erosion was slowed by TNF, IL-6 and IL-1 inhibitors, a JAK inhibitor, a CTLA4 agonist, and rituximab. Effects on bone mineral density varied between the biological DMARDs. Medications that are approved for the treatment of osteoporosis have been evaluated to prevent bone loss in rheumatic disease patients, including denosumab, cathepsin K, bisphosphonates, anti-sclerostin antibodies and parathyroid hormone (hPTH 1-34), and have some efficacy in both the prevention of systemic bone loss and reducing localized bone erosions. This article reviews the effects of biologic DMARDs on bone mass and erosions in patients with rheumatic diseases and trials of anti-osteoporotic medications in animal models and patients with rheumatic diseases.

Paradoxical side effects of bisphosphonates on the skeleton: What do we know and what can we do?

Bisphosphonates are considered the most effective drugs for controlling adult and pediatric osteolytic diseases. Although they have been used successfully for many years, several side effects, such as osteonecrosis of the jaw, delayed dental eruption, atypical femoral fracture, and alterations to the bone growth system, have been described. After an overview of nitrogenous bisphosphonate, the purpose of this article is to describe their mechanisms of action and current applications, review the preclinical and clinical evidence of their side effects in the skeleton ("what we know"), and describe current recommendations for preventing and managing these effects ("what we can do"). Finally, promising future directions on how to limit the occurrence of these side effects will be presented.

TIEG and estrogen modulate SOST expression in the murine skeleton.

TIEG knockout (KO) mice exhibit a female-specific osteopenic phenotype and altered expression of TIEG in humans is associated with osteoporosis. Gene expression profiling studies identified sclerostin as one of the most highly up-regulated transcripts in the long bones of TIEG KO mice relative to WT littermates suggesting that TIEG may regulate SOST expression. TIEG was shown to substantially suppress SOST promoter activity and the regulatory elements through which TIEG functions were identified using promoter deletion and chromatin immunoprecipitation assays. Knockdown of TIEG in IDG-SW3 osteocyte cells using shRNA and CRISPR-Cas9 technology resulted in increased SOST expression and delayed mineralization, mimicking the results obtained from TIEG KO mouse bones. Given that TIEG is an estrogen regulated gene, and as changes in the hormonal milieu affect SOST expression, we performed ovariectomy (OVX) and estrogen replacement therapy (ERT) studies in WT and TIEG KO mice followed by miRNA and mRNA sequencing of cortical and trabecular compartments of femurs. SOST expression levels were considerably higher in cortical bone compared to trabecular bone. In cortical bone, SOST expression was increased following OVX only in WT mice and was suppressed following ERT in both genotypes. In contrast, SOST expression in trabecular bone was decreased following OVX and significantly increased following ERT. Interestingly, a number of miRNAs that are predicted to target sclerostin exhibited inverse expression levels in response to OVX and ERT. These data implicate important roles for TIEG and estrogen-regulated miRNAs in modulating SOST expression in bone.

Differences between NMRI and DBA/2J mice in the development of somites and susceptibility to methylnitrosourea-induced skeleton anomalies.

The development of DBA/2J mouse strain embryos is nearly 12 h - or 6 somite pairs - delayed as compared to the outbred NMRI mouse embryos of the same age on gestation days (GD) 8-12. To evaluate inter-strain differences in susceptibility to teratogens, dams were treated with methylnitrosourea (MNU, 5 mg/kg body weight i.p.) on defined gestation days (NMRI: GD 9, 91/2 or 10; DBA/2J: GD 10 or 101/2). Skeletal anomalies produced by MNU on both mouse strains varied with the GD of treatment. The pattern of anomalies produced by MNU on a given GD markedly differed between the two mouse strains, yet they were similar -with a few exceptions- when exposures at equivalent embryonic stages are compared. Findings from this study indicated that strain-dependent differences in the developmental stage of mouse embryos of the same gestational age occur, a possibility that has been often neglected when inter-strain differences in susceptibility to developmental toxicants are interpreted.