Early morphological changes leading to central polydactyly, syndactyly, and central deficiencies: an experimental study in rats.

PURPOSE: Various combinations of central polydactyly, syndactyly, and cleft hand have been frequently observed in the individual hands and feet in the same patients. Little is known, however, about the early changes of abnormal induction of digital rays during limb development. To determine the early changes and process of formation of central polydactyly, syndactyly, and cleft hand, we experimentally induced these anomalies in the hind limbs of rat embryos and discussed the relationship among these abnormalities. METHODS: Inbred WKAH/Hkm rats were used for this study. Pregnant females were treated with busulfan at embryonic day (E) 11. The embryos were removed at E12 to E21 and stained with alcian blue and alizarin red S. The abnormal changes in the treated embryos' hind limbs were observed with a microscope. RESULTS: The edges of the footplates were irregular, and their growth was reduced at E14. By E15, abnormal clefts in the distal edge were present that disrupted the central digits (2 to 4) of the footplates. Because of these abnormal clefts, the digital rays were bent or branched, and the neighboring interdigital spaces were narrowed. These changes led to central polydactyly, syndactyly, and central deficiencies. CONCLUSIONS: These findings show that central polydactyly, syndactyly, and central deficiencies have the same early morphological changes: abnormal clefts in the central part of the footplate.

Busulfan-induced central polydactyly, syndactyly and cleft hand or foot: a common mechanism of disruption leads to divergent phenotypes.

The prevalence of clinical phenotypes that exhibit combinations of central polydactyly, syndactyly, or cleft hand or foot is higher than would be expected for random independent mutations. We have previously demonstrated that maternal ingestion of a chemotherapeutic agent, busulfan, at embryonic day 11 (E11) induces these defects in various combinations in rat embryo limbs. In an effort to determine the mechanism by which busulfan disrupts digital development, we examined cell death by Nile Blue staining and TdT-mediated dUTP nick end labeling (TUNEL) assays; we also carried out whole mount in situ hybridization for fibroblast growth factor-8 (Fgf8), bone morphogenetic protein-4 (Bmp4), and sonic hedgehog (Shh) to examine developmental pathways linked to these defects. In busulfan-treated embryos, diffuse cell death was evident in both ectoderm and mesoderm, peaking at E13. The increased cell death leads to regression of Fgf8 in the apical ectodermal ridge (AER) and Bmp4 and Shh in the underlying mesoderm. The subsequent pattern of interdigital apoptosis and cartilage condensation was variably disrupted. These results suggest that busulfan manifests its teratogenic effects by inducing cell death of both ectoderm and mesoderm, with an associated reduction in tissue and a disruption in the generation of patterning molecules during critical periods of digit specification.

Developmental abnormalities in rat embryos leading to tibial ray deficiencies induced by busulfan.

BACKGROUND: Little is known about the developmental changes associated with tibial ray deficiencies. The aim of this study was to detect cell death, proliferation, and gene expression that result in tibial ray deficiencies. METHODS: We induced tibial ray deficiencies in rat embryos using a teratogenic agent (busulfan) and observed the developmental changes in 1126 hindlimbs. We performed Nile blue staining, whole mount in situ hybridization for fibroblast growth factor 8 (Fgf8), bone morphogenetic protein 4 (Bmp4) and Sonic hedgehog (Shh), terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) and assessment of cell proliferation by 5-bromo-2'-deoxy-uridine (BrdU)/anti-BrdU immunohistochemistry. RESULTS: In situ hybridization showed reductions in Fgf8 and Bmp4 expression. Histological examination showed a delay of mesenchymal condensation, increased mesenchymal cell death, decreased mesenchymal cell proliferation, and a reduction in the number of mesenchymal cells. These abnormalities may cause hypoplasia of the limb. Bmp4 expression was markedly reduced in the anterior mesenchyme. Shh was expressed in the posterior mesenchyme. We suggest that the posterior skeletal elements may be fully formed owing to Shh expression, but the anterior skeletal elements may be underdeveloped owing to an intense reduction of Bmp4 expression in the anterior mesenchyme, causing hypoplasia of the tibial ray. CONCLUSIONS: The combined effects of increased cell death, decreased cell proliferation, reduction of Fgf8 expression, and intense reduction of Bmp4 expression in the anterior mesenchyme may play an important role in the development of tibial ray deficiency induced by busulfan.

Developmental failure of phalanges in the absence of growth/differentiation factor 5.

Growth/differentiation factor 5 (GDF5) is a member of the bone morphogenetic protein (BMP) family, which has been implicated in several skeletogenic events including cartilage and bone formation. To study the role of GDF5, we analyzed digit development in brachypodism (bp) mice, which carry functional null mutations of the Gdf5 gene and exhibit a reduction in the length of digit bones and loss of the middle phalanges. In situ detection of apoptosis and whole-mount detection of cell death showed abnormal apoptosis in the developing phalanges of bp mice. In situ hybridization in bp mice showed overexpression of Gdf5 mRNA in the developing phalanges where apoptotic cells were increased. In addition, bp mice exhibited excessive apoptosis in the interdigital regions. The condensed mesenchymal cells were progressively decreased in the developing phalanges and failed to form cartilage models of the middle phalanges. These findings show that excessive apoptosis in the absence of GDF5 results in developmental failure of the phalanges. We conclude that GDF5 is essential for maintenance and growth of the developing phalanges.

Matrix metalloproteinase-9 expression, tartrate-resistant acid phosphatase activity, and DNA fragmentation in vascular and cellular invasion into cartilage preceding primary endochondral ossification in long bones.

Vascular and cellular invasion into cartilage are essential for endochondral ossification. Recently it has been shown that matrix metalloproteinase-9 (MMP-9)/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. To study vascular and cellular invasion into cartilage preceding primary endochondral ossification in long bones, precursor femurs from 13- to 16-day-old murine embryos were sectioned. Tartrate-resistant acid phosphatase (TRAP) activity, in situ hybridization for matrix metalloproteinase-9 (MMP-9), immunostaining for CD31, and in situ detection of apoptosis (TUNEL) were studied. TRAP activity, MMP-9 mRNA, and CD31 expression were initially detected in the intertrabecular spaces of the perichondral collar, and then in cells migrating into the cartilage. The first cells involved in the primary invasion into cartilage were CD31-positive vascular endothelial cells and MMP-9-positive cells, followed by TRAP-positive cells. At the cartilage-marrow interface, CD31-positive vascular endothelial cells and MMP-9-positive cells were predominant. These results suggest that MMP-9-positive cells cooperate with vascular endothelial cells in cartilage angiogenesis. TUNEL-positive staining was detected on chondrocytes attached to the inner surface of the perichondral collar, and also detected in the area where cartilage was removed. These results suggest that chondrocytes separated from the cartilage matrix may undergo apoptosis.

Lmx1b expression during joint and tendon formation: localization and evaluation of potential downstream targets.

The tetrapod limb exhibits distinct dorsoventral joint, tendon, and muscle asymmetry. The LIM-homeodomain transcription factor, Lmx1b, is required to achieve the dorsal character of these structures, but the mechanism by which Lmx1b orchestrates this asymmetrical development is unknown. To identify target tissues and genes regulated by Lmx1b, we examined Lmx1b expression during joint, tendon and muscle formation (9.5-16.5 dpc) and the expression of several genes spatially restricted to developing joints and associated tissues in normal and Lmx1b knockout (KO) mice including: Gdf-5, sFrp2, sFrp3, Six1 and Six2. Lmx1b was diffusely expressed in the undifferentiated dorsal mesoderm of the emerging limb bud (E9.5-E11.5). With progressive proximal to distal differentiation, Lmx1b expression localized to dorsal joint-forming regions, to developing tendons and ligaments, but not to migrating myocytes (E13.5-15.5). By E16.5, mature tendon and ligament associations were evident and Lmx1b expression had regressed. The expression patterns of Gdf-5 and sFrp3 at E15.5 were symmetrical along the dorsoventral axis in normal and Lmx1b KO mice. sFrp2, Six1 and Six2 exhibited asymmetrical dorsoventral expression and in Lmx1b KO mice, this asymmetry is lost; however, none were solely restricted to or excluded from dorsal Lmx1b expressing tissues.

Involvement of the activin-follistatin system in tubular regeneration after renal ischemia in rats.

This study was conducted to investigate the involvement of the activin-follistatin system in renal regeneration after ischemic injury. Expression of mRNA for the activin beta(A) subunit was not detected in normal kidneys but increased markedly after renal ischemia. Immunoreactive beta(A) subunit was detected in tubular cells of the outer medulla in ischemic but not normal kidneys. Expression of mRNA for follistatin, an antagonist of activin A, was abundant in tubular cells of the outer medulla in normal kidneys and decreased significantly after renal ischemia. For assessment of the role of the activin-follistatin system in renal regeneration after ischemic injury, recombinant follistatin was intravenously infused into rats with renal ischemia, at the time of reperfusion. Exogenous follistatin prevented the histologic changes induced by ischemic injury, reduced apoptosis in tubular cells, and accelerated tubular cell proliferation. Serum levels of creatinine and blood urea nitrogen were significantly lower in follistatin-treated rats. Conversely, intravenous administration of recombinant activin A inhibited tubular cell proliferation after ischemic injury. These results indicate that the activin-follistatin system participates in renal regeneration after ischemic injury. Follistatin administered intravenously accelerates renal regeneration after renal ischemia, presumably by blocking the actions of endogenous activin.

Elevated levels of serum sulfite in patients with chronic renal failure.

Sulfite, a well known air pollutant, is toxic for humans, especially those with sulfite hypersensitivity. Sulfite is also generated endogenously, during normal metabolism of sulfur-containing amino acids. Mammalian tissues contain the enzyme sulfite oxidase, which detoxifies both endogenous and exogenous sulfite by oxidation to sulfate. Deficiency of sulfite oxidase in humans is fatal, demonstrating its physiologic importance. Nevertheless, information about serum and tissue levels of sulfite in normal and pathologic conditions is limited. Using a sensitive HPLC assay, it is shown here that sera from patients with chronic renal failure (CRF) contain significantly higher amounts of sulfite than those from healthy subjects. Mean +/- SD of serum sulfite in healthy subjects (n = 20) was 1.55 +/- 0.54 microM, whereas those in patients under maintenance hemodialysis (HD patients; n = 44) and CRF patients before introducing dialysis therapy (pre-HD patients; n = 33) were 3. 23 +/- 1.02 microM (P < 0.01) and 3.80 +/- 3.32 microM (P < 0.01), respectively. Among pre-HD patients, serum sulfite was positively correlated with serum creatinine (r = 0.714, P < 0.0001), and negatively with serum albumin (r = -0.407, P = 0.0188), hematocrit (r = -0.524, P = 0.0017), and total cholesterol (r = -0.375, P = 0. 0318). There was no significant association between sulfite and patient age, gender, or leukocyte counts. Multiple regression analysis revealed serum creatinine as the sole independent predictor of serum sulfite levels. Each HD treatment was associated with approximately 27% reduction in serum sulfite levels, suggesting the presence of a dialyzable form in serum. Thus, these results indicate that reduced glomerular filtration is a factor that determines serum sulfite levels. Chronic elevation in serum sulfite levels might contribute to tissue or organ dysfunction in patients with CRF.