Forelimb Buds Research Articles

Roles of retinoic acid receptors in early embryonic morphogenesis and hindbrain patterning.

Mutants mice carrying targeted inactivations of both retinoic acid receptor (RAR) alpha and RAR gamma (A alpha/A gamma mutants) were analyzed at different embryonic stages, in order to establish the timing of appearance of defects that we previously observed during the fetal period. We show that embryonic day (E)9.5 A alpha/A gamma embryos display severe malformations, similar to those already described in retinaldehyde dehydrogenase 2 null mutants. These malformations reflect early roles of retinoic acid signaling in axial rotation, segmentation and closure of the hindbrain; formation of otocysts, pharyngeal arches and forelimb buds; and in the closure of the primitive gut. The hindbrain of E8.5 A alpha/A gamma embryos shows a posterior expansion of rhombomere 3 and 4 (R3 and R4) markers, but fails to express kreisler, a normal marker of R5 and R6. This abnormal hindbrain phenotype is strikingly different from that of embryos lacking RAR alpha and RAR beta (A alpha/A beta mutants), in which we have previously shown that the territory corresponding to R5 and R6 is markedly enlarged. Administration of a pan-RAR antagonist at E8.0 to wild-type embryos cultured in vitro results in an A alpha/A beta-like hindbrain phenotype, whereas an earlier treatment at E7.0 yields an A alpha/A gamma-like phenotype. Altogether, our data suggest that RAR alpha and/or RAR gamma transduce the RA signal that is required first to specify the prospective R5/R6 territory, whereas RAR beta is subsequently involved in setting up the caudal boundary of this territory.

Comparison of the Embryotoxic Effects of Saporin, Agrostin (Type 1 Ribosome‐Inactivating Proteins) and Ricin (a Type 2 Ribosome‐Inactivating Protein

The effects of two type I ribosome-inactivating proteins, saporin and agrostin, and the type 2 ribosome-inactivating protein ricin and its constituent A and B chains, on the development of cultured mouse embryos, were investigated. Saporin and agrostin had similar embryotoxicity which was approximately 10(5) times weaker than that of ricin and its A and B chains, and the embryotoxicity of saporin and agrostin increased gradually with the dosage. In contrast, there was an abrupt rise in embryotoxictiy of ricin-A chain and B chain as the dose was raised from 10 to 20 ng x ml(-1). Saporin and agrostin did not affect the heart-beat and otic placode, but at 10 ng x ml(-1) ricin and 40 ng x ml(-1) ricin-A chain and B chain, all of the treated embryos exhibited abnormalities in heart-beat, yolk sac circulation, body axis, optic placode, otic placode, forelimb buds, branchial apparatus and cranial neural tube.

The Paralogous Hox Genes Hoxa10 and Hoxd10 Interact to Pattern the Mouse Hindlimb Peripheral Nervous System and Skeleton

The most 5′ mouse Hoxa and Hoxd genes, which occupy positions 9–13 and which are related to the Drosophila AbdB gene, are all active in patterning developing limbs. Inactivation of individual genes produces alterations in skeletal elements of both forelimb and hindlimb; inactivation of some of these genes also alters hindlimb innervation. Simultaneous inactivation of paralogous or nonparalogous Hoxa and Hoxd genes produces more widespread alterations, suggesting that combinatorial interactions between these genes are required for proper limb patterning. We have examined the effects of simultaneous inactivation of Hoxa10 and Hoxd10 on mouse hindlimb skeletal and nervous system development. These paralogous genes are expressed at lumbar and sacral levels of the developing neural tube and surrounding axial mesoderm as well as in developing forelimb and hindlimb buds. Double-mutant animals demonstrated impaired locomotor behavior and altered development of posterior vertebrae and hindlimb skeletal elements. Alterations in hindlimb innervation were also observed, including truncations and deletions of the tibial and peroneal nerves. Animals carrying fewer mutant alleles show similar, but less extreme phenotypes. These observations suggest that Hoxa10 and Hoxd10 coordinately regulate skeletal development and innervation of the hindlimb.

Rescuing the N-cadherin knockout by cardiac-specific expression of N- or E-cadherin.

Cell-cell adhesion mediated by some members of the cadherin family is essential for embryonic survival. The N-cadherin-null embryo dies during mid-gestation, with multiple developmental defects. We show that N-cadherin-null embryos expressing cadherins using muscle-specific promoters, alpha- or beta-myosin heavy chain, are partially rescued. Somewhat surprisingly, either N-cadherin or E-cadherin was effective in rescuing the embryos. The rescued embryos exhibited an increased number of somites, branchial arches and the presence of forelimb buds; however, in contrast, brain development was severely impaired. In rescued animals, the aberrant yolk sac morphology seen in N-cadherin-null embryos was corrected, demonstrating that this phenotype was secondary to the cardiac defect. Dye injection studies and analysis of chimeric animals that have both wild-type and N-cadherin-null cells support the conclusion that obstruction of the cardiac outflow tract represents a major defect that is likely to be the primary cause of pericardial swelling seen in null embryos. Although rescued embryos were more developed than null embryos, they were smaller than wild-type embryos, even though the integrity of the cardiovascular system appeared normal. The smaller size of rescued embryos may be due, at least in part, to increased apoptosis observed in tissues not rescued by transgene expression, indicating that N-cadherin-mediated cell adhesion provides an essential survival signal for embryonic cells. Our data provide in vivo evidence that cadherin adhesion is essential for cell survival and for normal heart development. Our data also show that E-cadherin can functionally substitute for N-cadherin during cardiogenesis, suggesting a critical role for cadherin-mediated cell-cell adhesion, but not cadherin family member-specific signaling, at the looping stage of heart development.

Transforming Growth Factor α (TGFα) Modulates the Effects of Genomic Imprinting and Prolongs Development of Parthenogenetic Mouse Embryos

The effect of transforming growth factor α (TGFα) on the development of diploid parthenogenetic mouse embryos (CBA × C57BL/6)F1was studied. The embryos were in vitro treated with the TGFα at the morula stage. Upon reaching the blastocyst stage, each embryo was implanted into uterus of a pseudopregnant female. At a dose of 5 ng/ml, the TGFα was found to improve development of parthenogenetic embryos before implantation, increase significantly the number of developing blastocysts, and promote embryo implantation into uterus. After treatment with TGFα at a dose of 10 ng/ml, 4% of parthenogenetic embryos reached the stage of 30–45 somites and had forelimb and hindlimb buds; the crown rump length of the embryo size from vertex to sacrum was 2.0 to 3.8 mm. A well-developed placenta was observed in 6% of TGFα-treated parthenogenetic embryos that reached the somite stages. In the parthenogenetic embryos with the most prominent development (40–45 somites) treated with 10 ng/ml of TGFα, the placental diameter was 4.0 to 4.2 mm on day 12 of gestation, which is close to the placental size of the normal (fertilized) 11-day-old mouse embryos. Our results suggest that exogenous TGFα can modulate the effects of genomic imprinting significantly improving formation of trophoblast derivatives and promoting longer postimplantation development of parthenogenetic embryos.

Teratogenicity of Edoferon Kappa A, a Molecule Derived from Salicylate, in Cultured Rat Embryos: Differences from Salicylate and Interaction with Free Oxygen Radical Scavenging Enzymes

The effect of edoferon kappa A (E-KA), a non-specific immunomodulatory and anti-neoplastic chemical substance derived from the methyl form of salicylate (acetyl salicylic acid; ASA), on mammalian embryos was studied and compared to the effects of ASA. Rat embryos were cultured in vitro from 9.5 days of gestation for 48 h. E-KA (0.1-12.8 mg/ml) and ASA (0.1-0.6 mg/ml) were added to the whole rat serum. To investigate the interaction of these molecules with antioxidant agents, the lowest effective concentrations of E-KA (0.6 mg/ml) and ASA (0.3 mg/ml) for all parameters were added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 mumol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. E-KA and ASA decreased growth and development in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology (haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud). There were no statistically significant differences between the control and embryos grown in the presence of 0.1-0.4 mg/ml E-KA, although the effects of ASA started at a concentration of 0.2 mg/ml. Embryos showed significant growth retardation in all scoring criteria and severe malformations when 0.5-3.2 mg/ml E-KA and 0.3-0.6 mg/ml ASA were added. When SOD was added, there was a significant decrease in the incidence of malformations and growth and developmental parameters were increased but this decrease never reached the control level. We concluded that E-KA has direct toxic effects on the developing embryo but at much higher concentrations than ASA, and the teratogenic effects of these molecules might be related to free oxygen radicals.

Suppression of polydactyly of the Gli3 mutant (extra toes) by deltaEF1 homozygous mutation.

Digit patterning is established through multiple genetic interactions. Delta-crystallin enhancer/E2-box factor (deltaEF1) is a zinc finger and homeodomain containing repressor protein, and is expressed in the posterior half of the forelimb bud and in the entire hindlimb bud during the early stage of limb development. The 6EF1-deficient mutant mice display various skeletal abnormalities, among which inferior ossification and abnormal patterning of autopodial bones are similar to those observed in Hox and Gli gene mutants. Gli3 mutant mice, extra toes (Xt), exhibit pre-axial polydactyly losing the identity of digit I. It is demonstrated here that deltaEF1null(lacZ) homozygosity suppressed formation of the extra digit, uniquely of the hindlimb, in both Gli3XtJ heterozygous and homozygous mutants, but with no restoration of digit I identity. In Gli3XtJ mutants, the Hoxd13 expression domain was expanded more dramatically in homozygotes. In Gli3XtJ;deltaEF1null(lacZ) double homozygous mutants, Hoxd13 expression once expanded in Gli3XtJ homozygous mutant was reduced, more conspicuously in the hindlimbs, which may account for hindlimb-restricted suppression of formation of the extra digit. The data suggest the possibility that the extent of Hoxd13 expression along the distal margin of the limb bud is determinative in defining the digit number.

Protection by free oxygen radical scavenging enzymes against salicylate-induced embryonic malformations in vitro

Salicylates are among the oldest and most widely used drugs and are known to lead to foetal death, growth retardation and congenital abnormalities in experimental animals. In this study, the effects of acetyl salicylic acid (ASA), salicylic acid (SAL) and sodium salicylate (NaSAL) on early organogenesis and the interaction of these molecules with free radicals has been investigated. Postimplantation rat embryos were cultured in vitro from day 9.5 of gestation for 48 hr. ASA, SAL and NaSAL were added to whole rat serum at concentrations between 0.1 and 0.6 mg/ml. Also, the lowest effective concentration of ASA for all parameters (0.3 mg/ml) and the same concentration of NaSAL and SAL was added to the culture media in the presence of superoxide dismutase (SOD) (30 U/ml) or glutathione (0.5 μmol/ml). The growth and development of embryos was compared and each embryo was evaluated for the presence of any malformations. When compared to growth of control embryos, the salicylates decreased all growth and developmental parameters in a concentration-responsive manner. There was also a concentration-related increase in overall dysmorphology, including the incidence of haematoma in the yolk sac and neural system, open neural tube, abnormal tail torsion and the absence of fore limb bud. When SOD was added in the presence of ASA, growth and developmental parameters were improved and there was a significant decrease in the incidence of malformations. Addition of SOD also decreased the incidence of malformations in the presence of SAL, but did not effect the growth and developmental parameters of SAL and NaSAL. There was no significant difference between the embryos grown in the presence of these three molecules on the addition of glutathione. The effects of salicylates might involve free oxygen radicals by the non-enzymatic production of the highly teratogenic metabolites 2,3- and 2,5-dihydroxybenzoic acid. An enhanced production of these metabolites in embryonic tissues may be directly related to the increased risk of congenital malformations.

Targeted Misexpression of Constitutively Active BMP Receptor-IB Causes Bifurcation, Duplication, and Posterior Transformation of Digit in Mouse Limb

Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior–posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior–posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression.

Expression ofLbx1 involved in the hypaxial musculature formation of the mouse embryo

Somites are the source of hypaxial musculature including skeletal muscles of the limb, tongue, and trunk. To get insight into the function of mouse Lbx1 homeobox gene in early somitic mesoderm differentiation, in situ hybridization analyses were performed. At the 4-6 somite stage (8 dpc), Lbx1 was first expressed in the lateral portion of the epithelial somite and dermomyotomal epithelium. This was in contrast to the expression of myf-5 in the medial region of the somite. The lateral expression of Lbx1 in somitic mesoderm then occurred regionally along the anterior-posterior body axis. Later, at 10 dpc (stage 1 of limb bud development), Lbx1-positive migrating cells originated in the lateral dermomyotomal lips at occipital, forelimb, and hindlimb levels. They also expressed Pax-3 and c-met, known as markers of the migrating limb muscle precursor cells. In stage 4 hindlimb bud (11.5 dpc), the dorsal and ventral muscle precursor populations expressed Lbx1. In stage 8 forelimb buds (12.5 dpc), Lbx1 expression was reduced in the proximal muscle masses, where the high expression of myogenin accompanying muscle differentiation was detected. These results suggest that mouse Lbx1 might be involved in the commitment or determination of a muscle cell subpopulation during hypaxial musculature development. J. Exp. Zool. 286:270-279, 2000.

Developmental characterization and chromosomal mapping of a LacZ transgene expressed in the mouse apical ectodermal ridge.

The apical ectodermal ridge (AER) has an essential role in limb morphogenesis involving the specification of the proximal-distal axis of the limb. During the analysis of transgenic mice that harbor a LacZ transgene, we detected strong expression of beta-galactosidase within the AER of developing embryos. In this mouse line, called Z16, the bacterial LacZ gene is linked to a Herpes simplex virus immediate early promoter that is normally silent in mice. Embryos from other independent mouse lines harboring the same DNA construct exhibited no AER specific staining. Thus, it appears that the LacZ transgene in the Z16 line is expressed in the AER in response to regulatory influences from genomic DNA flanking the integration site. By fluorescent in situ hybridization, the transgene insertion site was mapped to chromosome 12. Hemizygous and homozygous transgenic mice appear normal and are fertile. AER specific beta-galactosidase staining was detected by 9.5 days post coitum in the forelimb and hindlimb bud. beta-galactosidase staining could be seen throughout the development of the limbs up to 14.5 days post coitum when expression was restricted to the distal-most regions of the digits of the hindlimbs. The loss of beta-galactosidase staining between digits correlated with the onset of programmed cell death, or apoptosis, in the digit interzones. LacZ expression in this transgenic line represents a useful marker for studying AER function in limb specification during mouse embryogenesis.

Disrupting the establishment of polarizing activity by teratogen exposure

Between days 9.5 and 10, the forelimb buds of developing murine embryos progress from stage 1 which are just beginning to express shh and whose posterior mesoderm has only weak polarizing activity to stage 2 limbs with a distinguishable shh expression domain and full polarizing activity. We find that exposure on day 9.5 to teratogens that induce the loss of posterior skeletal elements disrupts the polarizing activity of the stage 2 postaxial mesoderm and polarizing activity is not subsequently restored. The ontogeny of expression of the mesodermal markers shh, ptc, bmp2, and hoxd-12 and 13, as well as the ectodermal markers wnt7a, fgf4, fgf8, cx43, and p21 occurred normally in day 9.5 teratogen-exposed limb buds. At stage 3, the treated limb apical ectodermal ridge usually possessed no detectable abnormalities, but with continued outgrowth postaxial deficiencies became evident. Recombining control, stage matched limb bud ectoderm with treated mesoderm prior to ZPA grafting restored the duplicating activity of treated ZPA tissue. We conclude that in addition to shh an early ectoderm-dependent signal is required for the establishment of the mouse ZPA and that this factor is dependent on the posterior ectoderm.

RYBP, a new repressor protein that interacts with components of the mammalian Polycomb complex, and with the transcription factor YY1.

The products of the Polycomb group (PcG) of genes are necessary for the maintenance of transcriptional repression of a number of important developmental genes, including the homeotic genes. A two-hybrid screen was used to search for putative new members of the PcG of genes in mammals. We have identified a new Zn finger protein, RYBP, which interacts directly with both Ring1 proteins (Ring1A and Ring1B) and with M33, two mutually interacting sets of proteins of the mammalian Polycomb complex. Ring1 binds RYBP and M33 through the same C-terminal domain, whereas the RYBP-M33 interaction takes place through an M33 domain not involved in Ring1 binding. RYBP also interacts directly with YY1, a transcription factor partially related to the product of the Drosophila pleiohomeotic gene. In addition, we show here that RYBP acts as a transcriptional repressor in transiently transfected cells. Finally, RYBP shows a dynamic expression pattern during embryogenesis which initially overlaps partially that of Ring1A in the central nervous system, and later becomes ubiquitous. Taken together, these data suggest that RYBP may play a relevant role in PcG function in mammals.

Hox C6 expression during development and regeneration of forelimbs in larval Notophthalmus viridescens.

A central theme concerning the epimorphic regenerative potential of urodele amphibian appendages is that limb regeneration in the adult parallels larval limb development. Results of previous research have led to the suggestion that homeobox containing genes are "re-expressed" during the epimorphic regeneration of forelimbs of adult Notophthalmus viridescens in patterns which retrace larval limb development. However, to date no literature exists concerning expression patterns of any homeobox containing genes during larval development of this species. The lack of such information has been a hindrance in exploring the similarities as well as differences which exist between limb regeneration in adults and limb development in larvae. Here we report the first such results of the localization of Hox C6 (formerly, NvHBox-1) in developing and regenerating forelimbs of N. viridescens larvae as demonstrated by whole-mount in situ hybridization. Inasmuch as the pattern of Hox C6 expression is similar in developing forelimb buds of larvae and epimorphically regenerating forelimb blastemata of both adults and larvae, our results support the paradigm that epimorphic regeneration in adult newts parallels larval forelimb development. However, in contrast with observations which document the presence of Hox C6 in both intact, as well as regenerating hindlimbs and tails of adult newts, our results reveal no such Hox C6 expression during larval development of hindlimbs or the tail. As such, our findings indicate that critical differences in larval hindlimb and tail development versus adult expression patterns of this gene in these two appendages may be due primarily to differences in gene regulation as opposed to gene function. Thus, the apparent ability of urodeles to regulate genes in such a highly co-ordinated fashion so as to replace lost, differentiated, appendicular structures in adult animals may assist, at least in part, in better elucidating the phenomenon of epimorphic regeneration.

The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity.

During embryonic development, initially similar fields can develop into distinct structures, such as the vertebrate fore- and hindlimbs. Although considerable progress has been made in our understanding of the genetic control underlying the establishment of the different limb axes, the molecular cues that specify the differential development of the fore- and hindlimbs are unknown. Possible candidates for genes determining limb identity are Pitx1, a gene whose transcripts are detected in the early hind- but not forelimb bud, and two members of the T-box (Tbx) gene family, Tbx4 and Tbx5, which are specifically expressed in the hindlimb and forelimb buds, respectively. Here we show that Tbx4 and Tbx5 are essential regulators of limb outgrowth whose roles seem to be tightly linked to the activity of three signalling proteins that are required for limb outgrowth and patterning: fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Wnt. In addition, we provide evidence that Tbx4 and Tbx5 are involved in controlling limb identity. Our findings provide insight into how similar developmental fields can evolve into homologous but distinct structures.

Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity.

In spite of recent breakthroughs in understanding limb patterning, the genetic factors determining the differences between the forelimb and the hindlimb have not been understood. The genes Pitx1 and Tbx4 encode transcription factors that are expressed throughout the developing hindlimb but not forelimb buds. Misexpression of Pitx1 in the chick wing bud induced distal expression of Tbx4, as well as HoxC10 and HoxC11, which are normally restricted to hindlimb expression domains. Wing buds in which Pitx1 was misexpressed developed into limbs with some morphological characteristics of hindlimbs: the flexure was altered to that normally observed in legs, the digits were more toe-like in their relative size and shape, and the muscle pattern was transformed to that of a leg.

Retinoid-induced limb defects 2: involvement of TGF-β2 in retinoid-induced inhibition of limb bud development

We previously demonstrated that retinoid-induced inhibition of chondrogenesis in the forelimb bud may be mediated by TGF-β2 (1). The present study was conducted to examine whether TGF-β2 is involved in the inhibition of forelimb bud development caused by all- trans -retinoic acid (RA). Expression of TGF-β2 was examined immunohistochemically in forelimb buds of embryos 24 h after dosing to the mother on Day 12 of gestation in the rat. In the control and 50 mg/kg group, TGF-β2 was expressed in the epithelium and prechondrogenic area around dead cells in the forelimb bud. In the 100 mg/kg group, a dose at which RA caused reduction defects of forearm bones, TGF-β2 expression was observed in the distal margin of forelimb buds, in which no expression was observed in the control and 50 mg/kg group. Immunohistologic studies also indicated that in the 100 mg/kg group, the expression of TGF-β2 was enhanced in forearm-bone prechondrocytes around the dead cells. In a whole embryo culture system, exposure to RA for 24 h reduced the proximodistal length and protein content in forelimb buds at concentrations of 3 μg/mL or more. The whole embryo culture system also showed that the expression of TGF-β2 was induced at the concentration of 3 μg/mL in the same region as found in forelimb buds of embryos from dams administered a teratogenic dosage of RA in vivo. Local application of TGF-β2 to the distal margin of the forelimb bud in Day 12 embryos reduced proximodistal growth and protein content in forelimb buds for 24 h in culture even without RA treatment. We also found that exogenous TGF-β2 inhibited DNA synthesis of forelimb bud cells in culture in a concentration-dependent manner. Neutralization of TGF-β2 with its antibody in the distal margin of forelimb buds partially prevented the RA-induced inhibition of forelimb bud growth in the whole embryo culture system. These results suggest that RA-induced TGF-β2 in the distal margin of forelimb buds may be involved in RA-induced inhibition of forelimb bud growth via reduction of cell proliferation in the distal margin, and RA-induced TGF-β2 in the prechondrogenic area may inhibit chondrogenesis in the future forearm bones, followed by reduction defects of the forearm bones.

Retinoid-induced limb defects 1: inhibition of cell proliferation in distal mesenchyme of limb buds in rats

The present study was undertaken to investigate the effects of all- trans -retinoic acid (RA) on cell death and limb bud growth in forelimb buds and also to examine whether these events are involved in limb bone defects induced by RA in rats. RA was given at doses of 50 and 100 mg/kg to pregnant rats on Day 12 of pregnancy. Although RA did not show teratogenecity in the 50 mg/kg group, micromelia was observed in the 100 mg/kg group in all live fetuses on Day 21 of gestation. Micromelia was characterized by high incidences of proximodistal reduction of forearm bones without reduction of the humerus. The incidence of cell death in prechondrogenic areas, which differentiate into humerus and forearm bone, significantly increased 24 h after RA treatment in not only the 100 mg/kg, but also the 50 mg/kg, group. There was no difference in the incidence of cell death in the prechondrogenic area between the two groups. These observations indicate that the bone-specific defects were not the result of cell death alone in the prechondrogenic area. We examined the effects of RA on early forelimb bud growth, which is indispensable for the morphogenesis of the forelimb. Proximodistal length and protein content were decreased significantly in the forelimb bud 24 h after RA treatment at a dose of 100 mg/kg, but not 50 mg/kg. The immunohistochemical detection of bromodeoxyuridine (BrdU) incorporated into cells showed that at a dose of 100 mg/kg, cell proliferation was reduced in the distal mesenchyme, but not in the forearm-bone prechondrocytes of the forelimb bud. As the distal margin provides the cells differentiating into the prechondrocytes of future bones in the limb bud, these observations suggested that RA-induced inhibition of cell proliferation in the distal margin resulted in a decrease of forearm-bone prechondrocytes localized at more distal sites. We conclude that RA may inhibit the chondrogenesis of forearm bones by reducing cell proliferation in the distal margin of the forelimb bud, not by increasing cell death, and that this results in reduction defects in forearm bones.

Expression of glucose-regulated proteins (GRP78 and GRP94) in hearts and fore-limb buds of mouse embryos exposed to hypoglycemia in vitro.

Hypoglycemia, the classic inducer of glucose-related protein (GRP) synthesis, is dysmorphogenic in rodent embryos and detrimentally affects the heart. This study compares GRP induction in a target vs non-target tissue by evaluating GRP expression in hearts and fore-limb buds of mouse embryos following exposure to hypoglycemia in vitro. Gestational day 9.5 embryos were exposed to 2, 6, and 24 h of either mild (80 mg/dl glucose) or severe (40 mg/dl glucose) hypoglycemia using the method of whole-embryo culture. GRP78 increased in a dose- and time-dependent fashion in embryonic hearts exposed to either 40 mg/dl or 80 mg/dl glucose, whereas GRP94 levels increased in hearts only after 24 h of hypoglycemia. In contrast to the heart, GRP induction in fore-limb buds occurred only with GRP78 following the most severe level and duration of hypoglycemia. RT-PCR analysis demonstrated an elevation in GRP78 and GRP94 message levels in embryonic hearts following severe hypoglycemia. However, mRNA levels did not increase in response to mild hypoglycemia. Overall, these data demonstrate the preferential induction of GRPs in the heart as compared to fore-limb buds in mouse embryos exposed to hypoglycemia. Increases in GRP protein levels may be a more reliable biomarker of stress than message levels. However, both tissues and methods should be examined for enhanced biomarker sensitivity.

Radiation-Induced Apoptosis and Limb Teratogenesis in Embryonic Mice

In utero irradiation of the fetus during the period of organogenesis induces a dramatic increase in malformation. However, the mechanisms underlying the teratogenesis remain to be elucidated. In the present study, the correlation between radiation-induced apoptosis and limb malformation was examined in mice. The mice were exposed to X rays in utero on day 11 of gestation during the period of organogenesis of limb buds. A marked increase in the number of apoptotic cells in the predigital regions in the forelimb buds was detected 4 h after irradiation. The preinterdigital regions of the forelimb buds did not show such an increase at the same time. Aphlangy and ectrodactyly were the main types of anomalies observed on day 19 in the limbs of the fetuses irradiated with 5 Gy. The increases in prenatal death and teratogenesis in limb digits in living fetuses were dependent on dose. The possible mechanisms involved are discussed.