Developmental Dynamics

doi: 10.1002/aja.1001930401pmid: N/A

Catlin, E. A.; Ezzell, R. M.; Donahoe, P. K.; Manganaro, T. F.; Ebb, R. G.; MacLaughlin, D. T.

doi: 10.1002/aja.1001930402pmid: 1511169

Mullerian inhibiting substance (MIS) is a 140,000 Mr Sertoli cell derived glycoprotein with a critical regulatory role in the male fetus initiated presumably by ligand binding with receptor. To localize this binding species we performed time course incubations of cultured fetal rat lungs or control tissues with MIS, applied rabbit anti‐MIS IgG, and fluorescein conjugated antirabbit IgG, and examined specimens with laser confocal microscopy. Punctate surface fluorescence followed by cytosolic and nuclear localization in lung consistent with specific adsorptive endocytosis was seen. Confocal imaging also detected MIS binding to the Mullerian duct in the urogenital ridge. Crosslinking of 125I‐MIS with plasma membranes revealed a high molecular mass binder with signal displaceable by excess unlabeled ligand. These data support the hypothesis that a specific plasma membrane binding protein for MIS exists. © 1992 Wiley‐Liss, Inc.

Sato, Tomotaro; Tuan, Rocky S.

doi: 10.1002/aja.1001930403pmid: 1511170

The developmental process of intramembranous ossification involves bone formation directly from mesenchymal differentiation without a cartilage intermediate. We have previously observed that systemic calcium deficiency in the developing chick embryo, produced by long‐term shell‐less culture, results in the appearance of chondrocyte‐like cells in the calvarium, a parietal bone which normally develops via intramembranous ossification. This investigation aims to analyze the mechanism underlying this calcium deficiency–related, aberrant appearance of cartilage phenotype in the chick embryonic calvarium. In view of the reported involvement of transforming growth factor β (TGF‐β) in osteogenesis and chondrogenesis, we have examined and compared here the expression of TGF‐β in the chick embryonic calvaria of normal (in ovo development, NL), shell‐less (SL), and calcium‐supplemented SL (SL + Ca) embryos. TGF‐β expression was analyzed at the mRNA level by blot and in situ cDNA hybridization, and at the protein level by immunohistochemistry and immunoblotting. The results presented here indicate that: (1) TGF‐β is expressed in the chick embryonic calvarium by both periosteal cells and osteocytes, as revealed by in situ hybridization and immunohistochemistry; (2) TGF‐β expression is significantly increased in SL calvarium compared to NL calvarium, at both protein and mRNA levels; (3) the number of TGF‐β expressing cells increases in the SL calvarium, particularly along the central, subcambial core region of the bone; and (4) exogenous calcium repletion to the SL embryo affects the expression of TGF‐β such that the pattern approaches that in the NL embryo. Taken together, these results indicate that altered TGF‐β expression accompanies the aberrant appearance of cartilage phenotype caused by systemic calcium deficiency. We postulate that normal cellular differentiation along the osteogenic pathway during embryonic intramembranous ossification is crucially dependent on regulated TGF‐β expression. © 1992 Wiley‐Liss, Inc.

Isokawa, Keitaro; Krug, Edward L.; Fallon, John F.; Markwald, Roger R.

doi: 10.1002/aja.1001930404pmid: 1511171

Recent in situ hybridization studies have correlated expression of potential regulatory genes with pattern formation in limb bud mesoderm (Tabin: Cell 66:199–217, 1991); however, the mechanism(s) controlling their expression in mesoderm and their relevance to the establishment of a limb morphogenetic pattern remain unknown. One likely candidate for regulating patterning events in limb mesoderm is the apical ectodermal ridge, as its removal in ovo results in a graded truncation of limb skeletal elements in the proximal‐distal axis dependent upon the time of excision (Rowe and Fallon: J Embryol Exp Morph 68:1–7, 1982). In the present study, we investigate whether the hypothetical imprint of ridge ectoderm is retained in cultured mesoderm. Specifically, we sought to determine if a subpopulation of limb mesoderm that forms in collagen gel culture (Markwald et al: Anat Rec 226:91–107, 1990), retains any expression of “limbness” in the absence of limb ectoderm as characterized by the formation of a predictable number and distribution of limb‐like chondrogenic elements in comparison to the temporal and spatial relationships of the in situ proximal, hindlimb skeletal structures. Accordingly, explants of undissociated mesoderm from stage 18–22 chicken leg buds were cultured without ectoderm on collagen gel lattices and the central subpopulation of mesoderm was examined morphologically. We show that this central subset of mesoderm will form chondrogenic cells which were not expressed uniformly throughout the subset, but rather distinct nodules or elements of cartilage were elaborated. Moreover, the number of elements expressed by the central subset increased with the age of the mesoderm at the time of explantation; spatially and temporally, the sequence of elements that formed always proceeded from the proximal, anterior margin of the subset to its distal, posterior border. The shapes of the initial elements (designated I and II) resembled the forms of in situ proximal skeletal structures (girdle and femur‐like), whereas more distal elements (III‐V) were often fused and without structural similarity to in situ skeletal structures. When cultures were established from the posterior mesoderm of stage 19/20 or 21 mesoblasts, the frequency of element I formation was reduced approximately one‐half, whereas formation of more distal elements was unaffected. Conversely, element formation from the central subset established from isolated anterior mesoderm was virtually identical to intact mesoblasts, indicating a capacity to regulate for the loss of mesoderm as occurs in situ (Hampé: Archs Anat Microsc Morph Exp 48:345–378, 1959). We interpret these findings to mean that limb mesoderm which forms the central subset is not merely competent to express in the absence of continuous ectodermal co‐culture, a chondrogenic phenotype, but also retains intrinsic, limblike morphogenetic potential to form, regulate, and, to a variable extent, shape a reproducibly specific number of cartilage elements. Because the number of chondrogenic elements that formed in culture appeared stage‐specific, a role for the apical ridge in specifying chondrogenic elements prior to its removal is supported. Indeed, our temporal findings correlate closely with the predictable truncation of leg skeletal structures following removal of the apical ridge in ovo, indicating that the endogenous ectodermal influence upon limb mesodermal morphogenesis can be assayed in vitro without the requirement of their continuous co‐culture. © 1992 Wiley‐Liss, Inc.

Beaulieu, J.‐F.; Millane, G.; Calvert, R.

doi: 10.1002/aja.1001930405pmid: 1511172

Two monoclonal antibodies were prepared against the duodenal mucosa of fourday‐old mice (MIM‐1/39 and MIM 1/130). The expression of the antigens was associated with the crypts of the small and large intestine in the fetus and adult. MIM‐1/39 was present in epithelial cells of the intervillous areas in the small intestine at 17 and 18 days of gestation; afterwards its expression was detected only in crypt cells from birth to adulthood. Transition from the mouth of the crypts to intestinal villi was abrupt. Expression of MIM‐1/39 was first detected at time of birth in the colon: In the adult, only crypt cells expressed the antigen and goblet cells were negative. Antigen MIM‐1/130 was detected from 16 to 18 days of gestation in the small intestine, in the mesenchymal matrix lying under the intervillous epithelium. After birth, it was present in the pericryptal mesenchymal matrix. This antigen was also expressed at birth in the colon and remained in the pericryptal matrix in the adult. In vivo, multiple injections of an organic extract of rat amniotic fluid to mothers, starting at 14 days of gestation, induced a profound modulation in the pattern of expression of both antigens at 17 days of gestation: The pattern of expression was comparable to that observed at least 5 days after birth in untreated animals. The expression of both antigens before crypt appearance may reflect some molecular differentiation in preparation for the formation of crypts, while their association with differentiated crypts may indicate that they have a role in the maintenance of crypt functional and/or morphological integrity. Finally, the fact that their expression can be modulated experimentally may prove to be a breakthrough for the study of crypt formation. © 1992 Wiley‐Liss, Inc.

Toyofuku, Toshihiko; Hoffman, John R.; Zak, Radovan; Carlson, Bruce M.

doi: 10.1002/aja.1001930406pmid: 1511173

The expression of α‐cardiac and α‐skeletal actin mRNA in regenerating muscle was examined. Changes in mRNA levels were analyzed in autografted extensor digitorum longus (EDL) muscles in rats using α‐isoform specific synthetic oligonucleotides and β‐actin cDNA as probes. After autografting, the expression of α‐cardiac actin mRNA was induced; concomitantly that of α‐skeletal actin mRNA was reduced. The pattern of α‐actin mRNA expression appeared to be similar to that seen in embryonic skeletal muscle. In order to evaluate the effects of innervation on α‐actin mRNA expression in regenerating muscle, nerveless, standard, and nerve‐intact autografted muscles were examined. More complete innervation facilitated the recovery of α‐skeletal actin mRNA to control levels, but had little effect on the amount of α‐cardiac actin mRNA. We found that regenerating muscle shows the embryonic pattern of α‐actin mRNAs in the early stage and concluded that the recovery of α‐skeletal actin mRNA expression to the adult pattern is influenced by innervation, while α‐cardiac actin mRNA expression is nerve independent. © 1992 Wiley‐Liss, Inc.

Potts, J. D.; Vincent, E. B.; Runyan, R. B.; Weeks, D. L.

doi: 10.1002/aja.1001930407pmid: 1511174

The formation of the valves in the heart is a spatially and temporally controlled process. A tissue interaction between the endothelium and its adjacent myocardium initiates the transformation of the endothelium into the mesenchymal precursors of the heart valve. One or more of the molecules implicated as critical for valve formation are members of the transforming growth factor β family of molecules. Presented here is a spatial and temporal analysis of TGFβ and TGFβ3 in the chick heart during valve formation. We show that TGFβ mRNA is concentrated in AV canal tissue where valve formation will occur, consistent with previous observations that TGFβ3 production is critical during valve formation. Additionally, an RNA complementary to TGFβ3 encoding mRNA is present in the heart. The temporally controlled appearance of RNA complementary to TGFβ3 suggests that this molecule may play a role in the regulation of TGFβ3 production in the heart. © 1992 Wiley‐Liss, Inc.

Corless, Christopher L.; Mendoza, Arturo; Collins, Tucker; Lawler, Jack

doi: 10.1002/aja.1001930408pmid: 1380845

Thrombospondin is an adhesive glycoprotein that is thought to play a role in tissue genesis and repair. We have used a monoclonal anti‐thrombospondin antibody, designated 5G11, to localize thrombospondin in paraformaldehyde fixed, paraffin‐embedded sections of developing mouse embryos. Thrombospondin expression is observed in uterine smooth muscle, endometrial glands, the decidua, and trophoblastic giant cells during the initial phase of post‐implantation development in the embryo. Cardiac myocytes and neuroepithelial cells show positive staining for thrombospondin at day 8.5 of gestation, and this expression continues throughout the development of the myocardium and central nervous system. Strong staining for thrombospondin is seen in developing bone and in the liver. Thrombospondin is also observed in developing smooth muscle and skeletal muscle, as well as in a variety of epithelia, including the epidermis, small intestinal epithelium, lens epithelium, renal tubular epithelium, and the epithelium of the developing tooth. Comparison of thrombospondin staining with that of two known cell surface receptors for thrombospondin, syndecan and the vitronectin receptor, reveals remarkable colocalization of thrombospondin and syndecan in all tissues, but almost no coexpression with the vitronectin receptor. Coexpression of thrombospondin and syndecan may play an important role in cell‐cell or cell‐matrix interactions during development. © 1992 Wiley‐Liss, Inc.

Yiping, Ling; Appelt, Denah; Kelly, Alan M.; Franzini‐Armstrong, Clara

doi: 10.1002/aja.1001930409pmid: 1511175

We have examined the histogenesis of the diaphragm and extensor digitorum muscle in rat embryos, with the aim of defining differences in developmental patterns that can be related to the functional requirements of these muscles during and after development. Patterns of interactions between myotubes and other cells, and frequency of gap junctions are quite different in the two muscles. In diaphragm, primary myotubes (at day 16 in utero) are closely associated with each other, forming parallel sheets or palisades and communicating by gap junctions. Secondary myotubes have formed by day 18, but are immature, and the frequency of gap junctions is lower. The arrangement in palisades is maintained even after fibers are separated from each other by their individual basal lamina. In EDL primary fibers at day 16 have fewer gap junctions, and the peak in communication occurs after the appearance of secondary myotubes (day 18 and 21). Secondary myotubes are more mature than in diaphragm at day 18. © 1992 Wiley‐Liss, Inc.

Gardner, Charles A.; Barald, Kate F.

doi: 10.1002/aja.1001930410pmid: 1354990

The protein products of both of the identified chick engrailed‐like (En) genes, chick En‐1 and chick En‐2, are localized in cells of the developing brain, mandibular arch, spinal cord, dermatome, and ventral limb bud ectoderm, as demonstrated by labeling with the polyclonal antiserum αEnhb‐1 developed by Davis et al. (Development 111:281–298, 1991). A subpopulation of cephalic neural crest cells is also En‐protein‐positive. The monoclonal antibody 4D9 recognizes the chick En‐2 gene product exclusively (Patel et al.: Cell 58:955–968, 1989; Davis et al., 1991) and colocalizes with chick En‐2 mRNA in the developing head region of the chick embryo as shown by in situ hybridization (Gardner et al.: J. Neurosci. Res. 21:426–437, 1988). In the present study we examine the pattern of αEnhb‐1 and 4D9 localization throughout the chick embryo from the first appearance of antibody (Ab)‐positive cells at stage 8 (Hamburger and Hamilton: J. Morphol. 88:49–92, 1951) through stage 28 (1–5.5 days). We compare the localization patterns of the two Abs to each other, as well as to the localization of the monoclonal Ab, HNK‐1, which recognizes many neural crest cells, using double‐and triple‐label fluorescence immunohistochemistry. Most En protein‐positive cells in the path of neural crest cell migration are not HNK‐1 positive. In detailed examination of αEnhb‐1 and 4D9 localization, we find previously undetected patterns of En protein localization in the prechordal plate, hindbrain, myotome, ventral body‐wall mesoderm, and extraembryonic membranes. Based upon these observations we propose: (1) that En expression in the mesoderm may be induced through interaction with En expressing cells in the neuroectoderm; (2) that En expression in the head mesenchyme is associated with somitomere 4; and (3) that En expression may be involved in epithelial‐mesenchymal cell transformations. © 1992 Wiley‐Liss, Inc.