Embryonic development of the coronal suture in man and rat.

Histological examination of skulls of 3- to 7-month-old human fetuses reveals in the fourth month of fetal life the development of a morphologically distinctive tissue in the area between the frontal and parietal bones. In this particular area, the pericranium tends to bulge outward while the dura mater shows the same in an inward direction. Consequently, a spindle-shaped blastema is enclosed. This spindle-like structure is only temporarily present; by about the sixth month of fetal life it has disappeared. Secondly, it was observed that prior to the development of this blastema mainly undifferentiated mesenchymal cells and only few fibroblasts are present in the area between the approaching ossification centres. In the full-grown blastema both types of cells are present in about equal numbers. However, once the blastema has been formed, fibroblasts predominate over mesenchymal cells. This tissue structure is also clearly distinguishable in the developing fetal rat skull. In this species, however, the spindle-shaped blastema appears at a later phase of fetal development, i.e. at about the 20th day. In both species, man and rat, the temporary presence of this blastema is not confined to the presumptive coronal suture only, but is demonstrable as well in other cranial sutures.     

Development of cartilage in transplanted future coronal sutures

From fetal rat skulls, tissue containing the 19-day-old presumptive coronal suture was excised and transplanted onto the exposed dura mater of adult rats. Host animals were sacrificed after 1, 2, 3, 4, 5 and 6 days. From the results of these experiments, the following conclusions can be drawn: (1) in all transplants chondrogenic activity occurred, resulting in the production of ectopic cartilage, and (2) cartilage development only starts on the cerebral side of the transplanted embryonic dura mater just beneath the area of the presumptive suture. Transplanted presumptive sutures of 21-day-old rats do not produce cartilage. The findings suggest that the suture undergoes a process of maturation. The existence of an osteogenesis-inhibiting mechanism, located in embryonic sutural tissue and being transmitted to the developing dura, is discussed.     

Basic fibroblast growth factor and transforming growth factor beta-1 expression in the developing dura mater correlates with calvarial bone formation

Numerous studies have found dura mater-calvarial mesenchyme interactions during calvarial bone induction; however, the exact molecular mechanisms governing these inductive events remain unknown. Recent studies have implicated basic fibroblast growth factor (FGF-2) and transforming growth factor-beta1 (TGF-beta1) in regulating bone formation. The purpose of this study was, therefore, to investigate the expression of FGF-2 and TGF-beta1 during calvarial bone formation in rats. Eight rats were killed on embryonic days 14, 18, and 20 and neonatal day 1 (n = 32). Four animals at each time point were analyzed by in situ hybridization, and the remainder were analyzed by immunohistochemistry. The results indicated that the dura mater underlying the developing calvarial bone strongly expressed FGF-2 and TGF-beta1 mRNA at all time points examined. In contrast, minimal growth factor expression was noted in the overlying calvarial mesenchyme until embryonic day 18, but it increased significantly with increasing age. Importantly, FGF-2 and TGF-beta1 mRNA expression in the dura mater underlying the developing calvarium preceded and was significantly greater than expression in the calvarium mesenchyme (p < 0.05). Interestingly, minimal expression of FGF-2 and TGF-beta1 mRNA was noted for all time points in the dura mater underlying the posterior frontal suture and within the posterior frontal suture connective tissue (p < 0.01 when compared with the dura mater underlying the developing calvarium). Immunohistochemical findings closely paralleled mRNA expression, with intense staining for FGF-2 and TGF-beta1 in the dura mater underlying the developing calvarial mesenchyme. Increasing FGF-2 and TGF-beta1 staining was noted within calvarial osteoblasts with increasing age, particularly in cells located near the endocranial surface (i.e., in contact with the developing dura mater). These findings, together with the known biologic functions of FGF-2 and TGF-beta1, implicate these growth factors in the regulation of calvarial bone growth by the developing dura mater. The possible mechanisms of this interaction are discussed.     

Nasal capsular cartilage is required for rat transpalatal suture morphogenesis

In the cranial vault, suture morphogenesis occurs when the growing cranial bones approximate and overlap or abut one another. Patency of developing sutures is regulated by the underlying dura mater. Once cranial sutures form, bone growth proceeds from the sutures in response to growth signals from the rapidly expanding neurocranium. Facial sutures do not develop in contact with the dura mater. It was therefore hypothesized that facial suture morphogenesis and bone growth from facial sutures are regulated by tissues with an equivalent role to the dura mater. The present study was designed to test this hypothesis by characterizing the morphology and growth factor expression in developing transpalatal (TP) sutures and their surrounding tissues, and then assessing the role of the overlying nasal capsular (NC) cartilages in maintaining suture patency. TP sutures develop as overlapping sutures, similar to cranial coronal sutures, and expression of Tgf-betas in TP sutures was similar to their distribution in cranial coronal sutures. To establish whether NC cartilages play a role in regulating TP suture morphogenesis, fetal rat TP sutures were cultured with associated attached NC cartilages or with NC cartilages removed. Sutures cultured for upward of 5 days with intact NC cartilages remained patent and maintained their cellular and fibrous components. However, in the absence of NC cartilages, the cellular nature of the sutures was not maintained and they became progressively acellular, with bony bridging across the suture. This finding is similar to that for cranial vault sutures cultured in the absence of dura mater, indicating that NC cartilages play an equivalent role to dura mater in maintaining the patency of developing sutures. These studies indicate that tissue interactions likely regulate morphogenesis of all cranial and facial sutures.     

A molecular analysis of the isolated rat posterior frontal and sagittal sutures: differences in gene expression

Although it is one of the most commonly occurring craniofacial congenital disabilities, craniosynostosis (the premature fusion of cranial sutures) is nearly impossible to prevent because the molecular mechanisms that regulate the process of cranial suture fusion remain largely unknown. Recent studies have implicated the dura mater in determining the fate of the overlying cranial suture; however, the molecular biology within the suture itself has not been sufficiently investigated. In the murine model of cranial suture fusion, the posterior frontal suture is programmed to begin fusing by postnatal day 12 in rats (day 25 in mice), reliably completing bony union by postnatal day 22 (day 45 in mice). In contrast, the sagittal suture remains patent throughout the life of the animal. Using this model, this study sought to examine for the first time what differences in gene expression--if any--exist between the two sutures with opposite fates. For each series of experiments, 35 to 40 posterior frontal and sagittal suture complexes were isolated from 6-day-old Sprague-Dawley rat pups. Suture-derived cell cultures were established, and ribonuicleic acid was derived from snap-frozen, isolated suture tissue. Results demonstrated that molecular differences between the posterior frontal and sagittal suture complexes were readily identified in vivo, although these distinctions were lost once the cells comprising the suture complex were cultured in vitro. Hypothetically, this change in gene expression resulted from the loss of the influence of the underlying dura mater. Significant differences in the expression of genes encoding extracellular matrix proteins existed in vivo between the posterior frontal and sagittal sutures. However, the production of the critical, regulatory cytokine transforming growth factor beta-1 was equal between the two suture complexes, lending further support to the hypothesis that dura mater regulates the fate of the overlying cranial suture.     

Cranial suture obliteration is induced by removal of transforming growth factor (TGF)-beta 3 activity and prevented by removal of TGF-beta 2 activity from fetal rat calvaria in vitro

Cranial suture morphogenesis requires soluble, heparin-binding factors secreted by the dura mater to resist premature osseous obliteration. Elevated levels of transforming growth factor (TGF)-beta 1, TGF-beta 2, and TGF-beta 3 have previously been noted in cranial sutures undergoing normal and premature sutural obliteration. To examine the role of TGF-beta s in regulating cranial suture morphogenesis, an established in vitro, serum-free, calvarial culture system was used. In this system, fetal rat coronal sutures undergo apparently normal suture morphogenesis in the presence of dura mater, but undergo osseous obliteration in the absence of dura mater. Neutralizing polyclonal antibodies to TGF-beta 1, TGF-beta 2, or TGF-beta 3 were added to cultures of fetal day 19 rat calvaria, which were harvested at 3, 4, or 5 days, processed for histology, sectioned, and examined. Coronal sutures from calvaria cultured in the presence of dura mater resisted obliteration, either alone or in the presence of TGF-beta 1 or TGF-beta 2 neutralizing antibodies. However, sutures from calvaria cultured in the presence of TGF-beta 3 neutralizing antibodies became obliterated. Conversely, sutures from calvaria cultured in the absence of dura mater became obliterated by bone, either alone or in the presence of neutralizing antibodies to TGF-beta 1 or TGF-beta 3. However, those sutures cultured in the presence of neutralizing antibodies to TGF-beta 2 were rescued from osseous obliteration.     

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.     

Expression and biological effect of urodele fibroblast growth factor 1: relationship to limb regeneration

Fibroblast growth factors (FGFs) have been previously implicated in urodele limb regeneration. Here, we examined expression of FGF-1 by blastema cells and neurons and investigated its involvement in wound epithelial formation and function and in the trophic effect of nerves. Neurons innervating the limb and blastema cells in vivo and in vitro expressed the FGF-1 gene. The peptide was present in blastemas in vivo. Wound epithelium thickened when recombinant newt FGF-1 was provided on heparin-coated beads, demonstrating that the FGF-1 was biologically active and that the wound epithelium is a possible target tissue of FGF. FGF-1 did not stimulate accessory limb formation. FGF-1 was as effective as 10% fetal bovine serum in maintaining proliferative activity of blastema cells in vitro but was unable to maintain growth of denervated, nerve-dependent stage blastemas when provided on beads or by injection. FGF-1 had a strong stimulating effect on blastema cell accumulation and proliferation of limbs inserted into the body cavity that were devoid of an apical epithelial cap (AEC). These results show that FGF-1 can signal wound epithelium cap formation and/or function and can stimulate mesenchyme accumulation/proliferation in the absence of the AEC but that FGF-1 is not directly involved in the neural effect on blastema growth.     

Development of interscapular brown adipose tissue in the hamster. II - Differentiation of transplants in the anterior chamber of the eye: role of the sympathetic innervation

The relationship between brown adipose tissue (BAT) and its sympathetic innervation during development was investigated by transplantation of undifferentiated (white fat-like) hamster BAT into the anterior eye chamber of adult hamsters. Such transplants are known to be revascularized and reinnervated by the vessels and the nerves of the host iris. The morphology of the BAT transplants was analysed during the post-operative weeks by light and electron microscopy, and the ingrowth of sympathetic nerve fibres from the iris was followed by radioautography. BAT appeared to differentiate in oculo, i.e. presented increasing amounts of adipocytes with multilocular fat deposits and abundant, well-developed mitochondria, but only after a delay of approx. 10 days, and remained much fatter than in situ. The establishment of the sympathetic innervation was not synchronous with the revascularization process. It occurred simultaneously with the morphological differentiation of the BAT transplants, and the nerve fibre density remained low. In the absence of sympathetic innervation, i.e. when the host irides were sympathectomized prior to transplantation, BAT still differentiated, but the process was further delayed and the proportion of differentiated brown adipocytes after 20 days in oculo was clearly lower than in control transplants. It is concluded that the sympathetic innervation in BAT is involved in the regulation of differentiating activity in the tissue, but is not obligatory for differentiation to occur.     

Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.     

Iron deficiency in the rat: biochemical studies of fetal metabolism

The effects of dietary-induced iron deficiency on fetal and maternal metabolism were studied in the rat. Concentrations of phenylalanine, but not tyrosine, were significantly elevated in plasma from iron-deficient maternal and fetal rats at day 20 of gestation with individual fetal plasma levels of phenylalanine as high as 10 mg per 100 ml. Concentrations of total 5-hydroxyindole compounds were significantly decreased in brain tissue from iron-deficient fetuses (day 20 of gestation), suggesting that synthesis of the compounds may be inhibited by iron deficiency. Mitochondrial NADH oxidase activity was markedly decreased (60%) in homogenates of fetuses at day 14 of gestation and may account for the high fetal resorption rate and small fetal size observed in the rat in iron deficiency.     

Responses to amputation of denervated ambystoma limbs containing aneurogenic limb grafts

The developing neural tubes and associated neural crest cells were removed from stage 30 Ambystoma maculatum embryos to obtain larvae with aneurogenic forelimbs. Forelimbs were allowed to develop to late 3 digit or early 4 digit stages. Limbs amputated through the mid radius-ulna regenerated typically in the aneurogenic condition. Experiments were designed to test whether grafts of aneurogenic limb tissues would rescue denervated host limb stumps into a regeneration response. In Experiment 1, aneurogenic limbs were removed at the body wall and grafted under the dorsal skin of the distal end of amputated forelimbs of control, normally innervated limbs of locally collected Ambystoma maculatum or axolotl (Ambystoma mexicanum) larvae. In Experiment 1, at the time of grafting or 1, 2, 3, 4, 5, 7, or 8 days after grafting, aneurogenic limbs were amputated level with the original host stump. At 7 and 8 days, this amputation included removing the host blastema adjacent to the graft. The host limb was denervated either one day after grafting or on the day of graft amputation. These chimeric limbs only infrequently exhibited delayed blastema formation. Thus, not only did the graft not rescue the host, denervated limb, but the aneurogenic limb tissues themselves could not mount a regeneration response. In Experiment 2, the grafted aneurogenic limb was amputated through its mid-stylopodium at 3, 4, 5, 7, or 8 days after grafting. By 7 and 8 days after grafting, the host limb stump exhibited blastema formation even with the graft extending out from under the dorsal skin. The host limb was denervated at the time of graft amputation. When graft limbs of Experiment 2 were amputated and host limbs were denervated on days 3, 4, or 5, host regeneration did not progress and graft regeneration did not occur. But, when graft limbs were amputated on days 7 or 8 with concomitant denervation of the host limb, regeneration of the host continued and graft regeneration occurred. Thus, regeneration of the graft was correlated with acquisition of nerve-independence by the host limb blastema. In Experiment 3, aneurogenic limbs were grafted with minimal injury to the dorsal skin of neurogenic hosts. When neurogenic host limbs were denervated and the aneurogenic limbs were amputated through the radius/ulna, regeneration of the aneurogenic limb occurred if the neurogenic limb host was not amputated, but did not occur if the neurogenic limb host was amputated. Results of Experiment 3 indicate that the inhibition of aneurogenic graft limb regeneration on a denervated host limb is correlated with substantial injury to the host limb. In Experiment 4, aneurogenic forelimbs were amputated through the mid-radius ulna and pieces of either peripheral nerve, muscle, blood vessel, or cartilage were grafted into the distal limb stump or under the body skin immediately adjacent to the limb at the body wall. In most cases, peripheral nerve inhibited regeneration, blood vessel tissue sometimes inhibited, but other tissues had no effect on regeneration. Taken together, the results suggest: (1) Aneurogenic limb tissues do not produce the neurotrophic factor and do not need it for regeneration, and (2) there is a regeneration-inhibiting factor produced by the nerve-dependent limb stump/blastema after denervation that prevents regeneration of aneurogenic limbs.     

Differentiation of embryonic hypothalamic transplants cultured on the choroidal pia in brains of adult rats

Summary Hypothalamic tissue from 16 to 18-day fetal rats was transplanted onto the choroidal pia overlying the superior colliculus in adult female rats. After survival periods of 2 weeks to 19 months, brains containing transplants were processed for monoamine fluorescence histochemistry, immunohistochemistry for three neuropeptides (LHRH, somatostatin, neurophysin), or for autoradiography in ovariectomized hosts that received [³H] estradiol. Most of the transplants survived and retained or increased in size; 14 of 25 transplants examined by fluorescence histochemistry were found to contain median eminence-like structures. In almost all of the transplants that were stained for neuropeptides, beaded processes and occasional cell bodies were observed. Although immunoreactive fibers were found near blood vessels, no palisade arrangement typical of the normal median eminence was evident. Each of the hypothalamic transplants on which steroid autoradiography was performed contained clusters of estrophilic neurons, the intensity of labeling of which was comparable to that seen in the host hypothalamus. These results indicate that many characteristic morphological and chemical features of the hypothalamus, which are not evident in the 16 to 18-day fetus, are elaborated in transplants during the survival period in the host. Transplantation of fetal hypothalamus to adult choroidal pia thus appears to be a valuable approach for studying the factors, humoral or neural, that regulate the differentiation of this brain region.     

Ontogeny of the response to growth hormone-releasing factor

Primary cell cultures were prepared from fetal, neonatal and adult rat pituitaries and evaluated for their ability to secrete growth hormone (GH) in response to growth hormone-releasing factor (GRF). Pituitary cells prepared from fetuses at days 19 and 21 of gestation, neonatal animals at the day of birth (day 0) or the following day (day 1) and peripubertal male rats showed full dose response curves to GRF with maximal GH release when stimulated with 1 X 10(-10) M rat GRF. At this concentration of GRF, the amount of GH released was not different from that elicited by activation of adenylate cyclase with 1 X 10(-5) M forskolin. In contradistinction, a preparation of cells from fetuses at day 18 of gestation did not show the same release of GH when challenged with 1 X 10(-10) M GRF and forskolin (0.057 +/- 0.001, compared to 0.076 +/- 0.003 micrograms/10(5) cells per 4.5 h), although the cells clearly responded to both secretagogues (basal levels of GH, 0.029 +/- 0.002 micrograms/10(5) cells per 4.5 h). While cells prepared from fetuses at day 21 of gestation or from animals after birth released 5-10% of their total cellular GH content, those prepared from 18- and 19-day fetuses released as much as 40% of their total GH suggesting there is a maturation of intracellular GH processing that occurs late in gestation. The results show that, in late pregnancy, the rat fetal pituitary is highly responsive to growth hormone-releasing factor and suggest that this peptide participates in regulating GH levels during the perinatal period.     

Craniosynostosis and altered patterns of fetal TGF-beta expression induced by intrauterine constraint

Recent work has demonstrated that fusion of the calvarial sutures is mediated by locally elaborated soluble growth factors, including the transforming growth factor-betas (TGF-betas), leading some to speculate that external biomechanical forces play little role in suture development. Clinical evidence has long suggested, however, that fetal head constraint may play a critical role in the pathogenesis of many cases of nonsyndromic craniosynostosis. The purpose of these experiments was to test the hypothesis that intrauterine constraint leads to an alteration in normal patterns of TGF-beta expression and that these alterations are associated with craniosynostosis. Fetal constraint was induced by allowing C57Bl/6 murine fetuses to grow for 2.5 days beyond the normal 20-day gestation by performing uterine cerclage on the eighteenth day. Cranial suture morphology was examined in hematoxylin and eosin-stained sections and in cleared whole-mount specimens, double stained with alizarin red S and Alcian blue. Expression patterns of TGF-beta1 and TGF-beta3 were examined by immunohistochemical techniques. Gross and microscopic examination of the cranial sutures of 17 constrained fetuses revealed changes that ranged from narrowing to complete osseous obliteration of the coronal and squamosal sutures. All sutures of 14 nonconstrained control pups remained patent. Fetal head constraint was associated with increased TGF-beta1 immunoreactivity within the new bone and the underlying dura when compared with nonconstrained age-matched controls. TGF-beta3 immunoreactivity was associated with the dura underlying patent, nonconstrained sutures, whereas constraint-induced synostosis was characterized by down-regulation of dural TGF-beta3 expression. These experiments confirm the ability of intrauterine constraint to induce premature fusion of the cranial sutures and provide evidence that intrauterine head constraint induces the expression of osteogenic growth factors in fetal calvarial bone and the underlying dura.     

Vasopressin gene expression is attenuated in the fetal Brattleboro rat

Due to a genetic defect the homozygous Brattleboro rat is unable to synthesize vasopressin gene products but still transcribes a mutant vasopressin mRNA from the gene. To study the influence of vasopressin gene products on the development of vasopressin gene expression, vasopressin mRNA levels of the supraoptic and paraventricular nucleus were measured at fetal day 20, postnatal day 1, 15 and 30 in the Wistar rat and in the heterozygous and homozygous Brattleboro rat by Northern blot analysis and in situ hybridization. In the homozygous Brattleboro rat of fetal day 20 and postnatal day 1, no or minute amounts of vasopressin mRNA were detectable but vasopressin mRNA was readily detectable at postnatal day 15 and 30. The Wistar rat and heterozygous Brattleboro rat had abundant vasopressin mRNA at fetal day 20 with increasing amounts towards postnatal day 30. The results indicate that vasopressin gene expression in the development of the homozygous Brattleboro rat is attenuated, possibly due to the absence of vasopressin gene products.     

Influence of endogenous growth hormone-releasing factor (GRF) on the secretion of GH during the perinatal period in the rat

Passive immunization of pregnant rats with a specific antiserum to rat GRF (GRF-AS) is followed by a decrease in fetal serum GH on the 19th day of gestation. A significant reduction in serum GH is still observed in older fetuses and newborn rats. Pituitary GH content increases in 19- and 20-day-old fetuses after GRF-AS administration to their mothers. These results suggest that endogenous fetal hypothalamic GRF (or placenta GRF) play a physiological role in the secretion of pituitary GH as early as the 19th day of fetal life and may be responsible for the peak of GH release that occurs in fetuses at the end of gestation.