Showing papers in "Developmental Dynamics in 1997"

Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis.

Abstract: To assess the role of the transcription factor Sox9 in cartilage formation we have compared the expression pattern of Sox9 and Col2a1 at various stages of mouse embryonic development. Expression of Col2a1 colocalized with expression of Sox9 in all chondroprogenitor cells. In the sclerotomal compartment of somites the onset of Sox9 expression preceded that of Col2a1. A perfect correlation was also seen between high levels of Sox9 expression and high levels of Col2a1 expression in chondrocytic cells. However, no Sox9 expression was detected in hypertrophic chondrocytes; only low levels of Col2a1 RNA were found in the upper hypertrophic zone. Coexpression of Sox9 and Col2a1 was also seen in the notochord. At E11.5 Sox9 expression in the brain and spinal neural tube was more widespread than that of Col2a1 although at E14.5 Sox9 and Col2a1 transcripts were colocalized in discrete areas of the brain. Distinct differences between Sox9 and Col2a1 expression were observed in the otic vesicle at E11.5. At E8.5, expression of Sox9 but not of Col2a1 was seen in the dorsal tips of the neural folds and after neural tube closure also in presumptive crest cells emigrating from the dorsal pole of the neural tube. No Col2a1 expression was detected in gonadal ridges in which high levels of Sox9 expression were detected. Together with our previous results showing that the chondrocyte-specific enhancer element of the Col2a1 gene is a direct target for Sox9, these results suggest that Sox9 plays a major role in expression of Col2a1. The correlation between high expression levels of Sox9 and high expression levels of Col2a1 in chondrocytes suggests the hypothesis that high levels of Sox9 are needed for full expression of the chondrocyte phenotype; lower levels of Sox9 such as in neuronal tissues which are also associated with lower expression levels of Col2a1 would be compatible with other cell specifications. Dev. Dyn. 209:377–386, 1997. © 1997 Wiley-Liss, Inc.

Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos

Abstract: BMP7 is expressed at diverse sites in the developing mouse embryo, including visceral endoderm, notochord, heart, eye, kidney, and bone. A null mutation in BMP7 results in defects largely confined to the developing kidney and eye. To examine whether other bone morphogenetic protein (BMP) family members potentially substitute for BMP7 in mutant embryos, thereby restricting the observed defects, we analyzed the expression patterns of BMP2 through BMP7 in wild-type and mutant tissues. In the central nervous system and heart, which develop normally in the absence of BMP7 signaling, expression domains of other BMP family members completely overlap with that of BMP7. The variable expressivity of the eye defect correlates with partially overlapping BMP4 and BMP7 expression domains during early eye induction. The loss of BMP7 signaling in the kidney results in apoptosis in the metanephric mesenchyme, a cell population that exclusively expresses BMP7. Thus, tissue defects observed in BMP7 deficient embryos are restricted to cell populations exclusively expressing BMP7. These data suggest that BMP family members can functionally substitute for BMP7 at sites where they colocalize in vivo.

Inactivation of two mouse alkaline phosphatase genes and establishment of a model of infantile hypophosphatasia

Abstract: We report the inactivation, via homologous recombination, of two of the three active mouse alkaline phosphatase genes, i.e., embryonic (EAP) and tissue nonspecific (TNAP). Whereas expression of the EAP isozyme was abolished in all tissues that express EAP developmentally (such as the preimplantation embryo, thymus, and testis), the EAP knock-out mice show no obvious phenotypic abnormalities. They reproduce normally and give birth to live offspring, indicating the nonessential role of EAP during embryonic development. Mice deficient in the TNAP gene mimic a severe form of hypophosphatasia. These TNAP-/- mice are growth impaired, develop epileptic seizures and apnea, and die before weaning. Examination of the tissues indicates abnormal bone mineralization and morphological changes in the osteoblasts, aberrant development of the lumbar nerve roots, disturbances in intestinal physiology, increased apoptosis in the thymus, and abnormal spleens. Our results indicate that, in the mouse, TNAP appears not to be essential for the initial events leading to bone mineral deposition but that TNAP seems to play a role in the maintenance of this process after birth. The other phenotypic manifestations may be a consequence of the lack of TNAP in the developing neural tube between stages E8.5 and E13.5 of embryogenesis. We hypothesize that the autonomic nervous system is compromised in these TNAP-/- mice.

Expression patterns of bone morphogenetic proteins (bmps) in the developing mouse tooth suggest roles in morphogenesis and cell differentiation

Abstract: Bone morphogenetic proteins (BMP) are secretory signal molecules which have a variety of regulatory functions during morphogenesis and cell differentiation. Teeth are typical examples of vertebrate organs in which development is controlled by sequential and reciprocal signaling between the epithelium and mesenchyme. In addition, tooth development is characterized by formation of mineralized tissues: the bone-like dentin and cementum as well as epithelially derived enamel. We have performed a comparative in situ hybridization analysis of the expression of six different Bmps (Bmp-2 to Bmp-7) starting from initiation of tooth development to completion of crown morphogenesis when dentine and enamel matrices are being deposited. Bmps-2, -4, and -7 were frequently codistributed and showed marked associations with epithelial-mesenchymal interactions. Their expression shifted between the epithelium and mesenchyme starting from the stage of tooth initiation. They were subsequently expressed in the enamel knot, the putative signaling center regulating tooth shape. Their expression domains prior to and during the differentiation of the dentine-forming odontoblasts and enamel-forming ameloblasts was in line with functions in regulation of cell differentiation and/or secretory activities of the cells. The expression of Bmp-3 was confined to mesenchymal cells, in particular to the dental follicle cells which give rise to the cementoblasts, forming the hard tissue covering the roots of teeth. Bmp-5 was expressed only in the epithelial ameloblasts. It was upregulated as the cells started to polarize and intense expression continued in the secretory ameloblasts. Bmp-6 was expressed only weakly in the dental mesenchyme during bud and cap stages. Our results are in line with regulatory functions of Bmps at all stages of tooth morphogenesis. Bmps-2, -4, and -7 are conceivably parts of signaling networks regulating tooth initiation and shape development. They as well as Bmp-5 may be involved in the induction and formation of dentine and enamel, and Bmp-3 in the development of cementum. The remarkable overlaps in the expression domains of different Bmp genes may implicate functional redundancy and/or formation of active heterodimers between different BMPs. Dev. Dyn. 1997;210:383–396. © 1997 Wiley-Liss, Inc.

Collagenase-3 (MMP-13) is expressed by hypertrophic chondrocytes, periosteal cells, and osteoblasts during human fetal bone development.

Abstract: Collagenase-3 (MMP-13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP-13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP-13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of 15 weeks. By in situ hybridization MMP-13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP-13 could be detected in osteoclasts. Furthermore, expression of MMP-13 mRNA was detected in osteoblasts and fibroblasts primarily on the inner side of calvarial bone of the skull at 16 weeks of gestation. Expression of MMP-13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming growth factor-beta (TGF-beta) and inhibited by bone morphogenetic protein-2 (BMP-2). No expression of MMP-13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP-13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification.

GAL4 enhancer traps expressed in the embryo, larval brain, imaginal discs, and ovary of Drosophila

Abstract: We have screened a collection of approximately 400 GAL4 enhancer trap lines for useful patterns of expression in the embryo, larval brain, imaginal discs, and ovary using a UAS-lacZ reporter construct. Although similar patterns of expression have previously been reported in the original P[lacZ] enhancer trap screens, these lines are useful for directing ectopic expression of genes in discrete patterns during these stages. In addition, we have identified some unique patterns of expression that have not been previously reported.

Dynamic expression of chicken sox2 and sox3 genes in ectoderm induced to form neural tissue

Abstract: The chick genes, cSox2 and cSox3, are members of a large family of genes that encode transcription factors. Previous studies have shown that these genes are predominantly expressed in the central nervous system during embryonic development. We show that cSox3 is expressed throughout the ectoderm that is compe- tent to form nervous tissue before neural induc- tion. The expression of cSox3 is lost from cells as they undergo gastrulation to form nonectoder- mal tissues; the transcription factor, Brachyury, appears in cells about to undergo gastrulation a short time before cSox3 transcripts are lost. Therefore, Brachyury expression may act func- tionally upstream ofcSox3downregulation.cSox3 expression is also lost from non-neuronal ecto- derm shortly after the neural plate becomes mor- phologically apparent.cSox2expression increases dramatically in the central nervous system as neural ectoderm is established. The appearance ofcSox2in neural ectoderm represents one of the earliest molecular responses to neural induction documented thus far. Dev. Dyn. 209:323-332, 1997. r 1997 Wiley-Liss, Inc.

Chemotaxis of skeletal muscle satellite cells

Abstract: Migration of myogenic cells occurs extensively during both embryogenesis and regeneration of skeletal muscle and is important in myoblast gene therapy, but little is known about factors that promote chemotaxis of these cells. We have used satellite cells from adult rats purified by Percoll density gradient centrifugation to test growth factors and wound fluids for chemotactic activity in blind-well Boyden chambers. Of a variety of growth factors tested only hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) exhibited significant chemotactic activity. The dose-response curves for both of these factors was bell-shaped with maximum activity in the 1-10 ng/ml range. Checkerboard analysis of TGF-beta showed that chemotaxis occurred only in response to a positive concentration gradient. An extract of rat platelets also exhibited chemotactic activity for satellite cells. Half-maximal activity of this material was about 3 micrograms/ml, and there was no evidence of inhibition of migration at high concentrations. Checkerboard analysis of platelet extract exhibited evidence of both chemotaxis and chemokinesis, or increase in random motility of cells. Inhibition experiments showed that most, but not all, of the chemotactic activity in platelet extract could be blocked with a neutralizing antibody to TGF-beta. A saline extract of crushed muscle was found to contain both mitogenic and motogenic factors for satellite cells. The two activities were present in different fractions after heparin affinity chromatography. We propose that the proliferation and migration of satellite cells during regeneration is regulated by overlapping gradients of several effector molecules released at the site of muscle injury. These molecules may also be useful for enhancing the dispersion of injected myoblasts during gene therapy.

Dlx genes encode DNA-binding proteins that are expressed in an overlapping and sequential pattern during basal ganglia differentiation.

Abstract: The Dlx gene family encodes homeodomain proteins that are required for forebrain and craniofacial development. Towards elucidating the roles for each of these genes, we have isolated cDNA clones encoding the full-coding sequence for murine Dlx-5 and partial coding sequence for murine Dlx-6. Three different classes of sense Dlx-5 cDNA clones were characterized, two of which lack the homeobox. We also identified an antisense Dlx-6 transcript. Genomic analysis shows that the Dlx-5 and -6 genes are linked. Biochemical analysis using gel shift assays demonstrate that DLX-1, -2 and -5 have very similar DNA-binding properties. The expression of Dlx-1, -2, -5, -6 and antisense Dlx-1 and -6 was studied in the midgestation mouse brain. We found that the Dlx genes are expressed in overlapping patterns at different stages of differentiation within the primordia of the basal ganglia. Dlx-1 and -2 are expressed in the least mature cells (in the ventricular and subventricular zones). Dlx-5 appears to be co-expressed with Dlx-1 and -2 in the SVZ, but is also expressed in the postmitotic cells of the mantle. Dlx-6 expression is strongest in the mantle. Antisense Dlx-1 and -6 have their highest expression in the SVZ. These results suggest that each of these Dlx genes may have a distinct role in different steps of differentiation in the basal ganglia.

Each member of the Id gene family exhibits a unique expression pattern in mouse gastrulation and neurogenesis

Abstract: We have performed a detailed comparative in situ hybridization analysis to examine the patterns of expression of all the members of the Id gene family (Id1–4) during marine gastrulation and neurogenesis. During gastrulation, both Id1 and Id3 are expressed in the tissues derived from the inner cell mass from 5.5 dpc onward, whereas Id2 is expressed in tissues derived from trophoblasts. Id4 expression is absent during this period of development. Embryonic Id1 messages are detected during gastrulation on the proximal side of the embryonic ectoderm, which is the border between the embryo proper and the extraembryonic tissues, and the expression of Id3 is found throughout the entire embryo proper. This unique pattern of expression of the different members of the Id family suggests a nonredundant role for these genes in antagonizing the activity of bHLH transcription factors during very early mouse development. During neurogenesis, the expression of each member of the Id family is present in an unique pattern along the dorsal-ventral axis of the neural tube: In the early stages of spinal cord development, both Id1 and Id2 are expressed in the roof plate, whereas Id3 is expressed both in the roof and the floor plates. As development progresses, the expression of both Id1 and Id3 is detected in the dividing neuroblasts, whereas Id2 and 4 are expressed in presumptive neurons which are undergoing maturation. The expression patterns of all the members of the Id gene family persist throughout the entire CNS, both in the spinal cord and in the brain. In addition, the characteristic expression of Id2 and Id4 in more mature neurons is reiterated both in the PNS and in the neurons of some of the sensory organs. These data suggest that the expression of different subgroups of the Id gene family may have different physiological consequences and thereby contributes in unique ways to specify the differentiation state of neuronal cells during development. Dev Dyn 208:92–106, 1997. © 1997 Wiley-Liss, Inc.

Expression Patterns of the Four Nuclear Factor I Genes During Mouse Embryogenesis Indicate a Potential Role in Development

Abstract: The nuclear factor I (NFI) fam- ily of site-specific DNA-binding proteins is re- quired for both the cell-type specific transcrip- tion of many viral and cellular genes and for the replication of adenovirus DNA.Although binding sites for NFI proteins within the promoters of several tissue-specific genes have been shown to be essential for their expression, it is unclear which NFI gene products function in specific tissues during development. We have isolated cDNAs from all four murine NFI genes (gene designations Nfia, Nfib, Nfic, and Nfix), assessed the embryonic and postnatal expression patterns of the NFI genes, and determined the ability of specific NFI proteins to activate transcription from the NFI-dependent mouse mammary tumor virus (MMTV) promoter. In adult mice, all four NFI genes are most highly expressed in lung, liver, heart, and other tissues but only weakly expressed in spleen and testis. The embryonic expression patterns of the NFI genes is complex, with NFI-Atranscripts appearing earliest—within 9 days postcoitum in the heart and developing brain. The four genes exhibit unique but over- lapping patterns of expression during embry- onic development, with high level expression of NFI-A, NFI-B, and NFI-X transcripts in neocortex and extensive expression of the four genes in muscle, connective tissue, liver, and other organ systems. The four NFI gene products studied differ in their ability to activate expression of the NFI-dependent MMTV promoter, with the NFI-B protein being most active and the NFI-A protein being least active. These data are dis- cussed in the context of the developmental expres- sion patterns of known NFI-responsive genes. The differential activation of an NFI-dependent promoter, together with the expression patterns observed for the four genes, indicate that the NFI proteins may play an important role in regulat- ing tissue-specific gene expression during mam- malian embryogenesis. Dev. Dyn. 208:313-325,

Trisomy eight in ES cells is a common potential problem in gene targeting and interferes with germ line transmission

Abstract: The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.

FGF-1 and FGF-7 induce distinct patterns of growth and differentiation in embryonic lung epithelium.

Abstract: Fibroblast growth factors (FGFs) and receptors (FGFRs) are expressed in the developing lung and appear to be major regulators of lung growth and differentiation. By using mesenchyme-free lung epithelial cultures we show that FGF-1 (aFGF) and FGF-7 (KGF) produce different effects in the developing lung. FGF-1 stimulates epithelial proliferation that results in bud formation (branching), while FGF-7 promotes epithelial proliferation that leads to formation of cyst-like structures. In addition, FGF-7 stimulates epithelial differentiation, stimulating expression of SP-A and SP-B mRNA throughout the explant, and inducing formation of focal areas of highly differentiated cells. The FGF-1 effects on differentiation are limited to induction of surfactant protein SP-B mRNA at the tips of the explant. The FGF-induced patterns of growth appear to correlate with the distribution of epithelial FGFRs mRNAs; FGFR-2 IIIb (KGFR) is diffusely expressed in the day 11 lung epithelium, while FGFR-4 appears in distal but not in proximal sites. We propose that cyst-like structures may result from FGF-7 binding to the uniformly distributed FGFR-2-IIIb. Lung bud formation may be regulated by FGF-1 and/or other ligands binding to FGFR-2 and a distally located FGFR, such as FGFR-4, leading to an increasing binding and activation of FGFRs at the tips of the explant. Thus, in the embryonic lung epithelium, growth effects of FGFs appear to be dependent on location of FGFRs, while effects on differentiation are ligand-dependent.

Developmental expression of perlecan during murine embryogenesis

Abstract: Perlecan is a modular heparan sulfate proteoglycan that is an intrinsic constitu- ent of all basement membranes and extracellular matrices. Because of its strategic position and unique structure, perlecan has been implicated in modulating the activity of various growth factors required for normal development and tissue homeostasis. To gain insights into the po- tential function of perlecan in vivo, we examined the spatiotemporal distribution of its mRNA and protein core during murine embryogenesis. We utilized a new affinity-purified antibody that rec- ognizes specifically the protein core of perlecan together with an in situ RT-PCR approach to perform a systematic analysis of perlecan expres- sion and deposition during murine ontogeny. Per- lecan appeared early (E10.5) in tissues of vasculo- genesis including heart, pericardium, and major blood vessels. Its early expression coincided with the development of the cardiovascular system. Subsequently (E11-13), the greatest deposition of perlecan occurred within the developing carti- lage, especially the cartilage undergoing endo- chondral ossification, where it remained ele- vated throughout all the developmental stages, and up to adulthood. Interestingly, the mRNA levels of perlecan were always higher in all the vascularized tissues, principally within endothe- lial cells, while chondrocytes displayed relatively low mRNA levels. This suggests a higher biosyn- thesis and turnover rates in the blood vessels vis-a ` -vis those of cartilaginous and other mesen- chymal tissues. During later stages of develop- ment (E13-17.5) perlecan mRNA levels progres- sively increased and its expression correlated with the onset of tissue differentiation of various parenchymal organs including the developing kidneys, lungs, liver, spleen, and gastrointestinal tract. The central nervous system showed no perlecan expression with the exception of the calvaria and choroid plexus. Collectively, the re- sults indicate that perlecan may play crucial roles not only in vasculogenesis but also in the maturation and maintenance of differentiated tissues, including cartilage. Dev. Dyn. 1997;210:

Tissue-specific expression of murine Nkx3.1 in the male urogenital system.

Abstract: The molecular mechanisms involved in growth and morphogenesis of the mammalian urogenital system are largely undefined. In this study, we describe the cloning and characterization of a novel murine homeobox gene, Nkx3.1, which is expressed in the male urogenital system during late embryogenesis and adulthood. We show that Nkx3.1 encodes a 38 kDa homeoprotein that has DNA binding properties similar to those of other Nkx family members. By RNAse protection analysis, we demonstrate that Nkx3.1 is expressed in late-gestation embryos and adults by tissues of the male urogenital system, including the testis, seminal vesicle, and the prostate. In adult males, expression of Nkx3.1 in the prostate increases during sexual maturation, and is significantly reduced following castration, suggesting that androgens are required for maintenance of Nkx3.1 expression. In situ hybridization analysis of mid- and late-gestation male embryos shows that Nkx3.1 is expressed in the developing urogenital sinus, testis, and prostatic buds. In addition to its expression in the urogenital system, we also find that Nkx3.1 is expressed in the dorsal aorta and kidney. These results implicate Nkx3.1 in the growth and development of the prostate and/or other tissues of the male urogenital system, and suggest that Nkx3.1 may play a role in sexually dimorphic as well as non-sexually dimorphic organogenesis.

Family of Ebf/Olf-1-related genes potentially involved in neuronal differentiation and regional specification in the central nervous system

Abstract: Two novel mouse genes, Ebf2 and Ebf3, have been identified which show high similarity to the rodent Ebf/Olf-1 and the Drosophila collier genes. The strong conservation of the protein regions corresponding to the DNA binding and dimerisation domains previously defined in Ebf strongly suggests that Ebf2 and Ebf3 also constitute DNA sequence-specific transcription factors. Determination of the chromosomal locations of the two genes indicated that the different members of this novel mouse multigene family are not clustered. A detailed analysis of the expression of each of the three Ebf genes in the developing central nervous system revealed partially overlapping patterns with two salient features: 1) In the region extending from the midbrain to the spinal cord, the expression of the three genes correlated with neuronal maturation, with a general activation in early post-mitotic cells, followed by specific patterns of extinction also consistent with the neurogenic gradient. 2) In the forebrain area, although the patterns of expression of the Ebf genes also reflected neuronal maturation, they appeared in addition to be region specific. These data suggest that Ebf genes may be involved in the control of neuronal differentiation in the CNS and in enforcing regional diversity in populations of post-mitotic forebrain neurons. Dev. Dyn. 1997;210:191–205. © 1997 Wiley-Liss, Inc.

Collagen fibrillogenesis in situ: Fibril segments become long fibrils as the developing tendon matures

Abstract: Tissue architecture, stability, and mechanical attributes are all determined by the structure and organization of collagen fibrils. Therefore, the characterization of fibril growth steps and determination of how this growth is regulated is essential to the elucidation of how tissues are assembled. We have proposed that fibril segments are intermediates in the formation of mature fibrils. The purpose of this study was to determine the length and structure of fibrils within a relatively mature tendon. The in situ determination of length performed here was only the second direct determination of fibril length in a vertebrate connective tissue and the first for a relatively mature tissue. The data demonstrate that the fibrils were discontinuous at 18 days of tendon development. However, both ends were not present in any of the analyzed fibrils within the 18-day tendon. Because the data set was 50-60 microm, this indicates a mean fibril length greater than 60 microm. These data are in contrast to data from the 14-day tendon, in which 80% of the fibrils had both ends in a 26-microm data set and the mean segment length was shown to be 10-30 microm. There were equal numbers of alpha and beta ends in the 18-day tendon. The structure of the ends was comparable to that in the less mature tendon. The data also indicate that fibril asymmetry and structure were maintained. The increase in fibril length is interpreted as being the result of a post-depositional, regulated assembly of segments via a lateral association/fusion to form mature fibrils. This hypothesis predicts an increase in diameter at this stage of development. The diameter increases have been documented, but this is the first demonstration of increases in length and maintenance of segment structure during this important stage of tendon development.

Transforming growth factor‐β3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane

Abstract: Studies on transforming growth factor β3 (TGF-β3) deficient mice have shown that TGF-β3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF-β3 in palatogenesis, we cultured TGF-β3 null mutant and wild-type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF-β3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF-β3 (−/−) samples is not impaired adhesion. Our data define a specific role for TGF-β3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane. Dev. Dyn. 209:255–260, 1997. © 1997 Wiley-Liss, Inc.

Comparative analysis of AP‐2α and AP‐2β gene expression during murine embryogenesis

Abstract: Transcription factor AP-2 has been identified as playing important roles during embryonic development of the neural tube, neural crest derivatives, skin, and urogenital tissues. Recently, we isolated a second AP-2 transcription factor, AP-2 beta, which is 76% homologous to the previously known AP-2 alpha gene, and showed that both genes are coexpressed in murine embryos at day 13.5 and 15.5 post coitum (pc). In the current study, we used specific cRNA probes to study comparatively AP-2 alpha and AP-2 beta expression by in situ hybridization of murine embryonic tissue sections. Our results reveal that expression of both genes starts at day 8 pc in the lateral head mesenchyme and extraembryonic trophoblast. The expression pattern was identical until day 10 pc but diverged significantly during later stages of development. From day 11 forward, specific expression patterns of AP-2 alpha and AP-2 beta mRNA were observed. Specific AP-2 beta signals were detected in the midbrain, sympathetic ganglia, adrenal medulla, and cornea. Specific AP-2 alpha signals were present in the limb buds, dorsal root ganglia, tooth germs, and Moll's and Meibom's glands. In contrast, expression of both genes occurred in skin, facial mesenchyme, spinal cord, cerebellum, and renal tubular epithelia. Our results indicate that both genes are expressed with different temporal and spatial patterns during embryonic development.

The Development of the Coronary Vessels and Their Differentiation Into Arteries and Veins in the Embryonic Quail Heart

Abstract: Research concerning the em- bryologic development of the coronary plexus has enriched our understanding of anomalous coronary vessel patterning. However, the differen- tiation of the coronary vessel plexus into arteries, veins, and a capillary network is still incomplete. Immunohistochemical techniques have been used for whole mounts and serial sections of quail embryo hearts to demonstrate endothelium, vas- cular smooth muscle cells, and fibroblasts. From HH35 onward, the lumen of the coronary plexus was visualized by injecting India ink into the aorta. In HH17, branches from the sinus venosus plexus expand into the proepicardial organ to reach the dorsal side of the atrioventricular sul- cus. From HH25 onward, vessel formation pro- ceeds toward the ventral side and the apex of the heart. After lumenized connections of the coro- nary vessels with the aorta and right atrium are established, a media composed of smooth muscle cells and an adventitia composed of procollagen- producingfibroblasts are formed around the coro- nary arteries. In the early stage, bloodflow through the coronary plexus is possible, although connections with the aorta have yet to be estab- lished. After the coronary plexus and the aorta and right atrium are interconnected, coronary vessel differentiation proceeds by media and ad- ventitia formation around the proximal coronary arteries. At the same time, the remodeling of the vascular plexus is manifested by disappearance of arteriovenous anastomoses, leaving only capil- laries to connect the arterial and venous system. Dev. Dyn. 208:338-348, 1997. r 1997 Wiley-Liss, Inc.

Differential expression of five laminin α (1-5) chains in developing and adult mouse kidney

Abstract: The nature of the laminin alpha chains in the embryonic and adult kidney is still being debated. The present study attempted to clarify this issue by immunofluorescence study using monoclonal antibodies against mouse alpha1, alpha2, and alpha5 chains and in situ hybridization for the alpha2, alpha3B, alpha4, and alpha5 mRNAs. Novel alpha1 chain-specific monoclonal antibodies against E8 fragment revealed a restricted distribution of alpha1 chain in a subset of epithelial basement membranes in the embryo, in agreement with previous mRNA data. The alpha2 mRNA was produced by mesenchyme, although the protein was deposited in epithelial basement membranes. The alpha3B mRNA was found only in a small subset of endothelial cells. The alpha4 mRNA was found transiently in embryonic mesenchyme, with particularly high levels in condensed mesenchyme, close to the tips of the ureteric tree where tubulogenesis is initiated. The alpha5 mRNA was strongly expressed by ureter epithelium but not expressed at early stages of tubulogenesis. Immunofluorescence verified low levels of the alpha5 chain in the early stages of tubulogenesis. However, during the capillary loop stage, the alpha5 chain became strongly expressed in the developing glomerular basement membrane, which matches the in situ hybridization results. During subsequent maturation of the kidney, the alpha5 chain became ubiquitously expressed in basement membranes. Overall, the alpha5 chain exhibited the broadest pattern of expression, followed by the alpha1 chain, particularly in the adult stage. These chains were the only ones produced by epithelial cells. Although some basement membranes contained several alpha chains, we failed to detect any of the five studied chains in some basement membranes. Thus, the identity of the alpha chains of many embryonic kidney blood vessels and several basement membranes in the inner medulla in the developing and adult kidney remain unclear.

Expression of reelin, the gene responsible for the reeler mutation, in embryonic development and adulthood in the mouse

Abstract: reelin has recently been isolated as a candidate gene, the mutation of which gives rise to the reeler phenotype in mice. In this study, we analyzed the expression of reelin during embryonic development in the mouse and in adult mouse tissues, by in situ hybridization. reelin transcripts were present on embryonic day (E) 8.5 in the somite, foregut, yolk sac, and unclosed neural plate. reelin was expressed in the brain, spinal cord, liver, and kidney throughout embryonic development, and transiently in many developing organs such as the optic cup, blood vessels, precartilage, stomach, pituitary, vibrissae, tooth germ, and in cells along growing nerve fibers. These observations indicate a role for reelin in development of organs in addition to that in neuronal migration. Furthermore, we demonstrated the existence of reelin mRNA and its cellular distribution in the adult brain, spinal cord, liver, kidney, testis, and ovary, suggesting additional roles for reelin in stabilizing the cyto-architecture and in remolding in adult organs. However, we detected no obvious phenotype of the reelin-expressing organs except for the brain in the reeler mouse, indicating the functional redundancy of this gene during the development of these organs.

Morphological analysis of germ cell apoptosis during postnatal testis development in normal and Hsp70‐2 knockout mice

Abstract: The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 26 and in juvenile Hsp 70-2 knockout mice. Adult Hsp 70-2 knockout males are infertile and lack spermatids and spermatozoa (Dix et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp 70-2 gene product (heat-shock protein 70-2 [HSP70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1;marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp 70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes.

Chondrocyte survival and differentiation in situ are integrin mediated.

Abstract: Chondrocytes in specific areas of the chick sternum have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix prior to bone deposition. Middle and caudal chondrocytes remain cartilaginous throughout development and continue to secrete collagen types II, IX, and XI. The interaction of integrin receptors with extracellular matrix molecules has been shown to affect cytoskeleton organization, proliferation, differentiation, and gene expression in other cell types. We hypothesized that chondrocyte survival and differentiation including the deposition into interstitial matrix of type X collagen may be integrin receptor mediated. To test this hypothesis, a serum-free organ culture sternal model that recapitulates normal development and maintains the three-dimensional relationships of the tissue was developed. We examined chondrocyte differentiation by five parameters: type X collagen deposition into interstitial matrix, sternal growth, actin distribution, cell shape, and cell diameter changes. Additional sterna were analyzed for apoptosis using a fragmented DNA assay. Sterna were organ cultured with blocking antibodies specific for integrin subunits (alpha2, alpha3, or beta1). In the presence of anti-beta1 integrin (25 microg/ml, clone W1B10), type X collagen deposition into interstitial matrix and sternal growth were significantly inhibited. In addition, all chondrocytes were significantly smaller, the actin was disrupted, and there was a significant increase in apoptosis throughout the specimens. Addition of anti-alpha2 (10 microg/ml, clone P1E6) or anti-alpha3 (10 microg/ml, clone P1B5) integrin partially inhibited type X collagen deposition into interstitial matrix; however, sternal growth and cell size were significantly decreased. These data are the first obtained from intact tissue and demonstrate that the interaction of chondrocytes with extracellular matrix is required for chondrocyte survival and differentiation.

Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: A role for transforming growth factor β3

Abstract: During early cardiac morphogenesis, outflow tract (OT) and atrio-ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle-like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor beta3 (TGF beta3) is an essential element in the progression of endothelial-transformation into mesenchyme. TGF beta(s) are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle-like cells. Using a monoclonal antibody against smooth muscle-specific alpha-actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGF beta3 initiates the expression of SMA, the pre-migratory AV endothelial monolayer was cultured with or without chicken recombinant TGF beta3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre-migratory endothelial cells cultured in complete medium (CM199) that contained TGF beta3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial-mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGF beta3. The induction of the expression of SMA by TGF beta3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial-mesenchymal transformation.

Basic fibroblast growth factor (FGF‐2) induced transdifferentiation of retinal pigment epithelium: Generation of retinal neurons and glia

Abstract: In the present study we report that basic fibroblast growth factor (bFGF, FGF-2) promotes the transdifferentiation ofXenopus lae- vis larval retinal pigment epithelium (RPE) into neural retina. Using specific antibodies we have examined the cellular composition of the regener- ated retinal tissue. Our results show that, in addition to retinal neurons and photoreceptors, glial cells were also regenerated from the transdif- ferentiated RPE. These results were specific to FGF-2,sinceotherfactorsthatweretested,includ- ing acidic FGF (aFGF, FGF-1), epidermal growth factor (EGF), laminin, ECL, and Matrigel, exhib- ited no activity in inducing retinal regeneration. These results are the first in amphibians demon- strating the functional role of FGF-2 in inducing RPE transdifferentiation. Transplantation stud- ies were carried out to investigate retinal regen- eration from the RPE in an in vivo environment. Sheets of RPE implanted into the lens-less eyes of larval hosts transformed into neurons and glial cells only when under the influence of host reti- nal factors. In contrast, no retinal transdifferen- tiation occurred if the RPE was implanted into the enucleated orbit. Taken together, these re- sults show that the amphibian RPE is capable of transdifferentiation into neuronal and glial cell- phenotypes and implicate FGF-2 as an important factor in inducing retinal regeneration in vitro. Dev. Dyn. 209:387-398, 1997. r 1997 Wiley-Liss, Inc.

Paired-related murine homeobox gene expressed in the developing sclerotome, kidney, and nervous system.

Abstract: We isolated a murine homeobox containing gene, Uncx41 The homeodomain sequence exhibits 88% identity to the unc-4 protein at the amino acid level In situ hybridization analysis revealed that Uncx41is expressed in the paraxial mesoderm, in the developing kidney, and central nervous system The most intriguing expression domain is the somite, where it is confined to the caudal part of the newly formed somite and subsequently restricted to the caudal domain of the developing sclerotome In the central nervous system, Uncx41is detected in the developing spinal cord, hindbrain, mesencephalon, and telencephalon The temporal and spatial expression pattern suggests that Uncx41 may play an important role in kidney development and in the differentiation of the sclerotome and the nervous system Dev Dyn 1997; 210:53–65 © 1997 Wiley-Liss, Inc

Agrin accumulates in the brain microvascular basal lamina during development of the blood-brain barrier

Abstract: The blood-brain barrier (BBB) is an essential feature of the microvasculature in neural tissues. Agrin, a synapse organizing molecule at the neuromuscular junction, also accumulates on brain microvasculature and may be involved in BBB formation and function. We investigated the developmental expression of agrin at rat and chick brain capillaries by immunohistochemistry and immunoblotting. Anti-agrin immunoreactivity (IR) completely ensheathes all observed microvessels labeled with anti-von Willebrand factor in adult brains of both species. Brain microvascular agrin IR codistributes with anti-laminin IR, consistent with agrin localization in the microvessel basal lamina. On microvessels in testis and thymus, tissues which also contain blood-tissue barriers, the pattern of IR is indistinguishable from brain microvessels. In contrast, little or no agrin IR is observed on capillaries in muscle and other tissues. During chick and rat development, agrin accumulates on brain microvessels around the time the vasculature becomes impermeable. Differential staining and electroblotting suggest that the agrin isoforms expressed on brain microvessels lack the 8- and 11-amino acid sequences that confer on agrin high potency in acetylcholine receptor clustering. Taken in context with the organizing role of agrin in synaptogenesis, these results indicate that agrins may function as important players in the formation and maintenance of cerebral microvascular impermeability.

Expression of the Sox11 gene in mouse embryos suggests roles in neuronal maturation and epithelio-mesenchymal induction

Abstract: Sry, the mammalian Y-linked testis determining gene, is a member of a family of genes known as Sox genes, which encode transcription factors related by a DNA-binding motif termed the HMG box. Sox genes are known to have diverse roles in vertebrate differentiation and development. We report here the cloning and characterisation of one of these genes, Sox11, in mice. In addition to an N-terminal HMG box domain, the deduced SOX11 protein contains a number of highly conserved C-terminal motifs, which may function in transcriptional regulation. Expression of Sox11 in mouse embryos was prominent in the periventricular cells of the central nervous system, suggesting a role in neuronal maturation. Expression was also observed in a wide range of tissues involved in epithelial-mesenchymal interactions, suggesting an additional role in tissue modelling during development.

Expression of ALK-1, a type 1 serine/threonine kinase receptor, coincides with sites of vasculogenesis and angiogenesis in early mouse development

Abstract: ALK-1 is a type I serine/threonine kinase receptor for members of the TGF-beta superfamily of growth factors; its endogenous ligand is not known. In this study, we have analyzed the temporal and spatial expression pattern of ALK-1 mRNA in mouse embryos from the one-cell zygote until 12.5 dpc using RT-PCR and in situ hybridization. ALK-1 mRNA was first detected in the embryo at 6.5 dpc. From 7.5-8.5 dpc expression was highest at sites of vasculogenesis in both the embryonic and extraembryonic part of the conceptus, in trophoblast giant cells, and in the endothelial lining of the blood vessels in the decidua. From 9.5-12.5 dpc, ALK-1 was found to be expressed in several different tissues and organs, but was highest in blood vessels, mesenchyme of the lung, submucosal layer of the stomach and intestines, and at specific sites of epithelial-mesenchymal interactions. Its expression pattern suggests that ALK-1 is a type I receptor for TGF-beta1 in the developing mouse.