Role of chondrogenic tissue in programmed cell death and BMP expression in chick limb buds

In the developing chick leg bud, massive programmed cell death occurs in the interdigital region. Previously, we reported the inhibition of cell death by separation of the interdigital region from neighboring digit cartilage. In this study, we examined the relationship between cell death and cartilaginous tissue in vitro. First, cell fate was observed with DiI that was used to examine cell movement in the distal tip of leg bud. Labeled cells in the prospective digital region were distributed only in the distal region as a narrow band, while cells in the prospective interdigital region expanded widely in the interdigit. In coculture of monolayer cells and a cell pellet tending to differentiate into cartilage, monolayer cells migrated into the cell pellet. These results suggested that digit cartilage tends to recruit neighboring cells into the cartilage during limb development. Next, we observed the relationship between cell death and chondrogenesis in monolayer culture. Apoptotic cell death that could be detected by TUNEL occurred in regions between cartilaginous nodules in mesenchymal cell culture. More apoptotic cell death was detected in the cell culture of leg bud mesenchyme of stage 25/26 than that of leg bud mesenchyme of stage 22 or that of stage 28. The most developed cartilaginous nodules were observed in the cell culture of stage 25/26. Finally, we observed Bmp expression in vitro and in vivo. Bmp-2, Bmp-4 and Bmp-7 were detected around the cartilage nodules. When the interdigit was separated from neighboring digit cartilage, Bmp-4 expression disappeared near the cut region but remained near the digit cartilage. This correlation between cell death and cartilaginous region suggests that cartilage tissue can induce apoptotic cell death in the developing chick limb bud due to cell migration accompanying chondrogenesis and Bmp expression.     

Essential mesenchymal role of small GTPase Rac1 in interdigital programmed cell death during limb development

Developing vertebrate limbs are often utilized as a model for studying pattern formation and morphogenetic cell death. Herein, we report that conditional deletion of Rac1, a member of the Rho family of proteins, in mouse limb bud mesenchyme led to skeletal deformities in the autopod and soft tissue syndactyly, with the latter caused by a complete absence of interdigital programmed cell death. Furthermore, the lack of interdigital programmed cell death and associated syndactyly was related to down-regulated gene expression of Bmp2, Bmp7, Msx1, and Msx2, which are known to promote apoptosis in the interdigital mesenchyme. Our findings from Rac1 conditional mutants indicate crucial roles for Rac1 in limb bud morphogenesis, especially interdigital programmed cell death.     

Activities of N-Myc in the developing limb link control of skeletal size with digit separation

The developing limb serves as a paradigm for studying pattern formation and morphogenetic cell death. Here, we show that conditional deletion of N-Myc (Mycn) in the developing mouse limb leads to uniformly small skeletal elements and profound soft-tissue syndactyly. The small skeletal elements are associated with decreased proliferation of limb bud mesenchyme and small cartilaginous condensations, and syndactyly is associated with a complete absence of interdigital cell death. Although Myc family proteins have pro-apoptotic activity, N-Myc is not expressed in interdigital cells undergoing programmed cell death. We provide evidence indicating that the lack of interdigital cell death and associated syndactyly is related to an absence of interdigital cells marked by expression of Fgfr2 and Msx2. Thus, instead of directly regulating interdigital cell death, we propose that N-Myc is required for the proper generation of undifferentiated mesenchymal cells that become localized to interdigital regions and trigger digit separation when eliminated by programmed cell death. Our results provide new insight into mechanisms that control limb development and suggest that defects in the formation of N-Myc-dependent interdigital tissue may be a root cause of common syndromic forms of syndactyly.     

The Cell Cycle and Cell Population Kinetics in the Programmed Cell Death in the Limb-Buds of Normal and 5-Bromodeoxyuridine- Treated Chick Embryos

The cell cycle and cell population kinetics have been analyzed in the interdigital regions of chick limb-buds during the course of programmed cell death both in normal and the 5-bromodeoxyuridine (BrdU)-treated embryos. Our previous study has shown that a single administration of BrdU at day 6 1/3 inhibited the programmed cell death occurring in normal development of limb-buds.
Pulse- as well as continuous labelings with tritiated thymidine (³H-TdR) were used. The results obtained from the analyses made on both normal and experimental embryos have demonstrated the presence of a particular DNA-synthetic period, around day 6 1/3, closely related to the programmed death occurring on day 7 1/3. In normal embryos, new cell populations, which did not belong to any phases of normal cell cycle, made their appearances in the process of programmed cell death. A possible correlation between programmed cell death and the cell cycle has been discussed in relation to the morphogenesis of limbs in both normal and BrdU-treated embryos.     

5-Aza-2'-deoxycytidine-induced cytotoxicity and limb reduction defects in the mouse

BACKGROUND: 5-Aza-2'-deoxycytidine (dAZA), causes hindlimb phocomelia in CD-1 mice. Studies in our laboratory have examined the hypothesis that compound- induced changes in gene expression may uniquely affect hindlimb pattern formation. The present study tests the hypothesis that dAZA causes limb dysplasia by inducing cytotoxicity among rapidly proliferating cells in the limb bud mesenchyme. METHODS: Pregnant CD-1 mice were given a teratogenic dose of dAZA (i.p.) at different times on GD 10 and fetuses evaluated for skeletal development in both sets of limbs by standard methods. Using general histology and BrdU immunohistochemistry, limb mesenchymal cell death and cell proliferation were then assessed in embryos at various times post dosing, shortly after initial limb bud outgrowth. The effect of dAZA on early limb chondrogenesis was also studied using Northern analysis of scleraxis and Alcian blue staining of whole mount limb buds. RESULTS: Compound related hindlimb defects were not restricted to a specific set of skeletal elements but consisted of a range of temporally related limb anomalies. Modest defects of the radius were observed as well. These results are consistent with a general insult to the limb mesenchyme. Mesenchymal cell death and reduced cell proliferation were also observed in both sets of limbs. The timing and location of these effects indicate a role for cytotoxicity in the etiology of dAZA induced limb defects. These effects also agree with the greater teratogenicity of dAZA in the hindlimb because they were more pronounced in that limb. The expression of scleraxis, a marker of early chondrogenesis, was reduced 12 hr after dAZA exposure, a time coincident with maximal cell death, as was the subsequent emergence of Alcian blue stained long bone anlagen. CONCLUSIONS: These findings support the hypothesis that cytotoxic changes in the limb bud mesenchyme during early limb outgrowth can induce the proximal limb truncations characteristic of phocomelia after dAZA administration.     

Effects of digit tissue on cell death and ectopic cartilage formation in the interdigital zone in chick leg buds

In developing chick leg buds, large-scale cell death occurs in the interdigital zone, which is responsible for the separation of digits from each other. Ectopic cartilage formation is known to occur upon removal of the chondrogenic digit tissue of the leg bud. To examine the mechanisms of ectopic cartilage formation in the interdigital cell death region, we performed the following operations on stage 28–29 leg buds: (i) removal of the digit-forming area; (ii) incision between the interdigital zone and digit region; (iii) insertion of an aluminum barrier into the interdigital zone; and (iv) insertion of a permeable Nuclepore filter into the interdigital zone. In all cases, the inhibition of cell death and/or the formation of ectopic cartilage in the interdigital zone were observed, although the frequency of the inhibition of cell death and the formation of ectopic cartilage varied, depending upon the position where the operations were performed. These results suggest that cell death and cell differentiation in the interdigital zone may be controlled by some factor(s) from digit cartilage.     

TGF-beta is required for programmed cell death in interdigital webs of the developing mouse limb

During limb formation massive cell death in the mesenchyme of the interdigital spaces accompanies the formation of free digits. Members of the transforming growth factor beta (TGF-) superfamily were discussed to play a key role in cell-cell interactions, important in the regulation of programmed cell death (PCD). TGF-beta itself is believed to be involved in epithelial-mesenchymal interactions. Here, we demonstrate that PCD is significantly reduced in interdigital spaces of the developing limbs of Tgfbeta2-/-Tgfbeta3-/- double knockouts. The regression of interdigital webs seems to be doses-dependent as interdigital mesenchyme is at least partly reduced in Tgfbeta2-/-Tgfbeta3+/- mutants, whereas interdigital zones of Tgfbeta2-/-Tgfbeta3-/- double knockouts reveal only minimal signs of regression. We conclude that TGF- is a critical extrinsic regulator of PCD.     

The role of the polarizing zone in the pattern of experimental chondrogenesis in the chick embryo interdigital space

We have previously shown that removal of the apical ectodermal ridge of the third interdigital space of the chick leg bud at stages 28 and 29 is followed by the appearance of ectopic cartilage, which in the course of development gives rise to extra digits. These in vivo studies suggest that the pattern of skeletal morphogenesis in the limb depends on the inhibitory effect of the ectoderm. In the present study we tested whether zone polarizing activity (ZPA) exerted an effect on the pattern of experimental chondrogenesis in the interdigital space of the leg bud in stage 29 HH chick embryos. A small fragment of tissue from the ZPA in chick embryos in which ZPA activity was most intense was grafted onto the interdigital space in which chondrogenesis had previously been experimentally induced. No significant changes were observed in the course of differentiation of the recipient interdigital spaces with ZPA grafts, leading us to conclude that the graft failed to modify the morphogenetic fate of interdigital tissue.     

The BMP antagonist Gremlin regulates outgrowth, chondrogenesis and programmed cell death in the developing limb.

In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.     

Tamoxifen-Dependent,Inducible Bmp2CreER Drives Selective Recombinase Activity in Early Interdigital Mesenchyme and Digit Collateral Ligaments

During limb development, the interdigital mesenchyme has been proposed to play a signaling role instructing morphogenesis of different digit types, as well as undergoing programmed cell death necessary to free digits in animals not adapted for swimming or flying. We have generated a conditional, tamoxifen-dependent Cre line, Bmp2CreER, which drives highly selective recombination restricted to the distal limb mesoderm, largely restricted to the interdigits, and selectively active in digit ligament but not tendon progenitors at later stages. The Bmp2CreER provides a valuable new tool to dissect roles of interdigital mesenchyme and potentially investigate divergence of ligament and tendon lineages.     

Sequential induction of syndecan, tenascin and cell proliferation associated with mesenchymal cell condensation during early tooth development

The cell surface proteoglycan, syndecan, and the extracellular matrix glycoprotein, tenascin, are expressed in the mesenchyme during early development of many organs. We have studied the expression patterns of syndecan and tenascin during initiation of tooth development and in association with mesenchymal cell condensation and compared these with cell proliferation. Syndecan, tenascin and bromodeoxyuridine (BrdU) incorporation were localized by triple-labelling immunohistochemistry in serial sections of molar tooth germs of mouse embryos. Prior to formation of the epithelial tooth bud, syndecan accumulated in the mesenchymal cells which underlie the presumptive dental epithelium, but tenascin was not detected at this stage. Tenascin appeared during initiation of the epithelial down-growth at the lingual aspect of the tooth germ. During subsequent formation of the epithelial bud, at the late bud stage, syndecan and tenascin became exactly colocalized in the condensed mesenchyme which was clearly demarcated from other jaw mesenchyme. The expression of syndecan and tenascin was accompanied by rapid cell proliferation as indicated by marked BrdU incorporation. When development advanced to the cap stage, syndecan staining intensity in the dental papilla mesenchyme increased further whereas tenascin became reduced. In conclusion, the results demonstrate that the expression patterns of syndecan and tenascin overlap transiently during the period of mesenchymal cell condensation and that this is accompanied by cell proliferation. Syndecan and tenascin may play a role in growth control and in compartmentalization of the dental mesenchymal cells in the condensate.     

Programmed cell death in the embryonic vertebrate limb

The developing limb bud provides one of the best examples in which programmed cell death exerts major morphogenetic functions. In this work, we revise the distribution and the developmental significance of cell death in the embryonic vertebrate limb and its control by the BMP signalling pathway. In addition, paying special attention to the interdigital apoptotic zones, we review current data concerning the intracellular death machinery implicated in mesodermal limb apoptosis.     

Role of FGFs in the control of programmed cell death during limb development

We have investigated the role of FGFs in the control of programmed cell death during limb development by analyzing the effects of increasing and blocking FGF signaling in the avian limb bud. BMPs are currently considered as the signals responsible for cell death. Here we show that FGF signaling is also necessary for apoptosis and that the establishment of the areas of cell death is regulated by the convergence of FGF- and BMP-mediated signaling pathways. As previously demonstrated, cell death is inhibited for short intervals (12 hours) after administration of FGFs. However, this initial inhibition is followed (24 hours) by a dramatic increase in cell death, which can be abolished by treatments with a BMP antagonist (Noggin or Gremlin). Conversely, blockage of FGF signaling by applying a specific FGF-inhibitor (SU5402) into the interdigital regions inhibits both physiological cell death and that mediated by exogenous BMPs. Furthermore, FGF receptors 1, 2 and 3 are expressed in the autopodial mesoderm during the regression of the interdigital tissue, and the expression of FGFR3 in the interdigital regions is regulated by FGFs and BMPs in the same fashion as apopotosis. Together our findings indicate that, in the absence of FGF signaling BMPs are not sufficient to trigger apoptosis in the developing limb. Although we provide evidence for a positive influence of FGFs on BMP gene expression, the physiological implication of FGFs in apoptosis appears to result from their requirement for the expression of genes of the apoptotic cascade. We have identified MSX2 and Snail as candidate genes associated with apoptosis the expression of which requires the combined action of FGFs and BMPs.     

Sequential changes of programmed cell death in developing fetal mouse limbs and its possible roles in limb morphogenesis

Apoptotic cell death in the developing limb of mouse fetuses was examined sequentially on days 11–15 of gestation by means of Nile blue (NB) sulfate staining with special reference to its relation to limb morphogenesis. With some exceptions, programmed cell death (PCD) in the hand and foot was observed in the mesenchyme but not in the surface ectoderm. We found that during digital formation PCD begins at the proximal portion of the interdigital mesenchyme and subsequently expands distally. Therefore, the initial PCD that occurs in the interdigital zones may determine the proximal ends of digital separation and also contribute to the demarcation between the palm (sole) and digits (toes). During digital separation, the areas of PCD in the interdigital zones were found to become larger and expand distally on day 13, which may be necessary for the separation of digits and for determining the interdigital area to disappear. PCD in presumptive phalangeal joints was also found to proceed from proximal to more distal joints. The PCD in presumptive joints may be required for the separation of phalanges and metacarpal (metatarsal) bones and for the formation of joint cavities. In addition, intense PCD was observed in the radial (tibial) and ulnar (fibular) margins of the hand and foot plates for 4–5 days. Such PCD at marginal areas seems to prevent the formation of supernumerary digits (preaxial and postaxial polydactyly) and other digital malformations. Therefore, the timing when PCD commences and ends, the sites where PCD occurs, and the intensity, duration, and proximo-distal progress of PCD appear to be genetically determined, and the elimination of unnecessary cells by PCD may be essential for normal limb morphogenesis. The present findings also suggest that the normal progress of PCD in the hand and foot plates of rodent fetuses may prevent the formation of some limb malformations such as webbing fusion of digits, polydactyly, or cleft hand/foot. © 1996 Wiley-Liss, Inc.     

Differential expression of two msh-related homeobox genes Chox-7 and Chox-8 during chick limb development.

We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.     

Coordinated and sequential activation of neutral and acidic DNases during interdigital cell death in the embryonic limb

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.     

BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling

In vertebrate limbs that lack webbing, the embryonic interdigit region is removed by programmed cell death (PCD). Established models suggest that bone morphogenetic proteins (BMPs) directly trigger such PCD, although no direct genetic evidence exists for this. Alternatively, BMPs might indirectly affect PCD by regulating fibroblast growth factors (FGFs), which act as cell survival factors. Here, we inactivated the mouse BMP receptor gene Bmpr1a specifically in the limb bud apical ectodermal ridge (AER), a source of FGF activity. Early inactivation completely prevents AER formation. However, inactivation after limb bud initiation causes an upregulation of two AER-FGFs, Fgf4 and Fgf8, and a loss of interdigital PCD leading to webbed limbs. To determine whether excess FGF signaling inhibits interdigit PCD in these Bmpr1a mutant limbs, we performed double and triple AER-specific inactivations of Bmpr1a, Fgf4 and Fgf8. Webbing persists in AER-specific inactivations of Bmpr1a and Fgf8 owing to elevated Fgf4 expression. Inactivation of Bmpr1a, Fgf8 and one copy of Fgf4 eliminates webbing. We conclude that during normal embryogenesis, BMP signaling to the AER indirectly regulates interdigit PCD by regulating AER-FGFs, which act as survival factors for the interdigit mesenchyme.