Extracellular matrix fluctuations during early embryogenesis.

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node).

Multi-field 3D scanning light microscopy of early embryogenesis.

A computer-controlled microscopy system was devised to allow the observation of avian embryo development over an extended time period. Parallel experiments, as well as extended specimen volumes, can be recorded at cellular resolution using a three-dimensional scanning procedure. The resulting large set of data is processed automatically into registered, focal- and positional-drift corrected mosaic images, assembled as montages of adjacent microscopic fields. The configuration of the incubator and a sterile embryo chamber prevents condensation of the humidified culturing atmosphere in the optical path and is compatible with both differential interference contrast and epifluorescence optics. As a demonstration, recordings are presented showing the large-scale remodelling of the embryonic primordial vascular structure.

Transient nuclear localization of Fyn kinase during development in zebrafish.

Fyn protein tyrosine kinase is present in the unfertilized and fertilized egg, becomes activated within minutes following fertilization, and has been localized to the cortical cytoplasm and spindle apparatus of the zygote. In order to establish the expression pattern of Fyn in the early embryo, we examined the distribution pattern of Fyn by immunofluorescence microscopy. Fyn protein is distributed evenly among cells of the cleavage stage zebrafish embryo and is concentrated in the cortical region of each cell. During blastula and gastrula stages, Fyn was expressed in all cells, however a subpopulation of cells exhibited strong nuclear staining for Fyn. Nuclear Fyn staining was not observed after the gastrula period of development, nor in the adult zebrafish. Immunoprecipitation of Fyn from isolated mid-blastula nuclei confirmed Fyn was present in the nucleus. This is, to our knowledge, the first demonstration of Fyn kinase, which lacks a nuclear localization signal, present in the nucleus. The transient compartmentalization of Fyn in the nucleus could be important in nuclear signaling.

Fertilization-induced activation of phospholipase C in the sea urchin egg.

Fertilization results in the biphasic activation of polyphosphoinositide-specific phospholipase C (PLC) activity with an initial increase in activity coincident with the sperm-induced calcium transient, followed by a more sustained increase prior to mitosis. Immunoprecipitation studies demonstrated that the gamma isoform of PLC is present in both the unfertilized and the fertilized egg and contributes to the initial phase of PLC activation. Fertilization also resulted in translocation of a significant fraction of PLC-gamma from the cytosol to the membrane compartment of the egg.

Identification of the developmental marker, JB3-antigen, as fibrillin-2 and its de novo organization into embryonic microfibrous arrays.

The monoclonal antibody JB3 was previously shown to react with a protein antigen present in the bilateral primitive heart-forming regions and septation-stage embryonic hearts; in addition, primary axial structures at primitive streak stages are JB3-immunopositive (Wunsch et al. [1994] Dev. Biol. 165:585-601). The JB3 antigen has an overlapping distribution pattern with fibrillin-1, and a similar molecular mass (Gallagher et al. [1993] Dev. Dyn. 196:70-78; Wunsch et al. [1994] Dev. Biol. 165:585-601). Here we present immunoblot and immunoprecipitation data showing that the JB3 antigen is secreted into tissue culture medium by day 10 chicken embryonic fibroblasts, from which it can be harvested using JB3-immunoaffinity chromatography. A single polypeptide (Mr = 350,000), which was not immunoreactive with an antibody to fibrillin-1, eluted from the affinity column. Mass spectroscopy peptide microsequencing determined the identity of the JB3 antigen to be an avian homologue of fibrillin-2. Live, whole-mounted, quail embryos were immunolabeled using a novel microinjection approach, and subsequently fixed. Laser scanning confocal microscopy indicated an elaborate scaffold of fibrillin-2 filaments encasing formed somites. At more caudal axial positions, discrete, punctate foci of immunofluorescent fibrillin-2 were observed; this pattern corresponded to the position of segmental plate mesoderm. Between segmental plate mesoderm and fully-formed somites, progressively longer filamentous assemblies of fibrillin-2 were observed, suggesting a developmental progression of fibrillin-2 fibril assembly across the somite-forming region of avian embryos. Extensive filaments of fibrillin-2 connect somites to the notochord. Similarly, fibrillin-2 connects the mesoderm associated with the anterior intestinal portal to the midline. Thus, fibrillin-2 fibrils are organized by a diverse group of cells of mesodermal or mesodermally derived mesenchymal origin. Fibrillin-2 microfilaments are assembled in a temporal and spatial pattern that is coincident with cranial-to-caudal segmentation, and regression of the anterior intestinal portal. Fibrillin-2 may function to impart physical stability to embryonic tissues during morphogenesis of the basic vertebrate body plan.

Relationship of the extracellular matrix to coronary neovascularization during development.

The main goal of this study was to determine the temporal and spatial relationship of several components of the extracellular matrix (ECM) to coronary vascularization during prenatal and early postnatal development. Rat microvessels were visualized by immunolabeling for platelet endothelial cell adhesion molecule (PECAM-1), and by exposure to the lectin from Griffonia simplicifolia I. Coronary vasculogenesis, which first occurs in gestation day 13 (E13) hearts, was preceded by the deposition of fibronectin. The onset of laminin immunoreactivity in basement membranes coincided with tube formation and was followed by the appearance of collagen IV. Discontinuous collagen IV staining of basement membranes typified early tube formation but progressed to completely encircle capillaries. Sparse staining of collagen I and III was observed in prenatal hearts, but increased after birth. Staining for both molecules was limited mainly to the adventitia of vessels larger than capillaries, and as a component of septa and the epicardium. To determine the effects of loading conditions on key ECM molecules relating to neovascularization, avascular E12 rat hearts were grafted to the anterior eye chamber of adult hosts. In these hearts, which are hemodynamically unloaded, the appearance and distribution of ECM components were similar to hearts developing in utero. It was concluded that during heart development: (1) fibronectin may provide a primary scaffolding for the migration of primordial endothelial cells/angioblasts; (2) tube formation coincides with lamin deposition and is closely followed by the appearance of collagen IV; (3) collagens I and III are not related to tube formation in the prenatal heart; and (4) the relationship of the ECM to vessel formation is not notably altered in the absence of a ventricular load. Furthermore the early onset of PECAM-1 immunoreactivity suggests that it is a useful endothelial marker and may play a role in tube formation.

Influence of graft innervation on neovascularization of embryonic heart tissue grafted in oculo.

We have shown that sympathetic denervation increases subendocardial capillarity during left ventricular hypertrophy. To determine the direct effects of sympathetic innervation on development of the coronary microvasculature in the absence of hemodynamic load, we grafted avascular fetal rat atrial or ventricular tissue into the anterior eye chamber of host rats which had undergone unilateral superior cervical gangliectomies. Innervation to the contralateral eye chamber remained intact. The grafts were harvested 14 or 35 days later, and volume densities of blood vessels, myocytes, and extracellular matrix were determined using standard point-counting techniques on low-power electron micrographs. Graft perfusion and metabolism were assessed simultaneously with thallium-201 and 2-[14C]deoxy-D-glucose uptake, respectively. Innervation did not significantly alter the vascular volume densities or cellular composition of atrial or ventricular tissue compared with noninnervated tissue after either 14 or 35 days in oculo. Similarly, innervation did not significantly alter graft perfusion or metabolism. We conclude that sympathetic innervation does not directly influence the growth of the microvasculature or myocardial metabolism in hemodynamically unloaded, developing heart tissue.

The extracellular matrix during heart development.

The embryonic extracellular matrix, which is comprised of glycosaminoglycans, glycoproteins, collagens, and proteoglycans, is believed to play multiple roles during heart morphogenesis. Some of these ECM components appear throughout development, however, certain molecules exhibit an interesting transient spatial and temporal distribution. Due to significant new data that have been gathered predominantly in the past 10 years, a comprehensive review of the literature is needed. The intent of this review is to highlight work that addresses mechanisms by which extracellular matrix influences vertebrate heart development.

Coronary neovascularization of embryonic rat hearts cultured in oculo is independent of thyroid hormones.

We tested the hypothesis that neovascularization of embryonic hearts is independent of thyroid hormones. To dissociate the effects of hemodynamic load from the direct influence of thyroid hormones on coronary neovascularization, we grafted avascular, gestational day 12 rat hearts to the anterior eye chamber of adult rats receiving daily injections of thyroxine (T4; 0.25 mg/kg sc), propylthiouracil (PTU; 10 mg/kg ip), or saline (control). After 21 days, grafts were perfuse fixed and prepared for transmission electron microscopy. Neither T4 nor PTU treatment affected size or mass of the unloaded graft, or the volume densities of vessels, myocytes, or the extracellular matrix, which were determined by standard point-counting techniques. Thus neither exogenous administration of T4 nor the induction of hypothyroidism in host rats produced a significant effect on the growth or vascularity of the unloaded hearts. We conclude that vascular growth in embryonic rat hearts cultured in oculo is independent of thyroid hormones and that the angiogenic effect of thyroid hormones in loaded hearts is a consequence of increased work-related events.

Neovascularization of embryonic rat hearts cultured in oculo closely mimics in utero coronary vessel development.

Coronary neovascularization was studied following grafting of avascular hearts from gestation day-12 (E-12) rat embryos to the anterior eye chambers of adult rats. Volume densities (Vv) of vessels, myocytes, and the extracellular matrix (ECM) after 3-7, 14, 21, and 35 days in oculo were compared to Vv in hearts developing in utero at E-15, E-18, and E-20. The myocardium in both models exhibited similar vessel Vv and capillary developmental stages: (1) clustering of endothelial cells and red blood cells; (2) endothelial cell migration, and (3) tube formation/maturation. The Vv of myocytes increased while that of the ECM remained constant over time. Cross-species grafting utilizing species-specific antibodies determined that the majority, but not all, of the 10-day graft vasculature was of graft origin. Therefore, both de novo growth (vasculogenesis) and sprouting (angiogenesis) were occurring in oculo. Tracer molecules infused into host rats reached the outermost graft vessels only after 10 days in oculo, suggesting a functional link with the host circulation after this time. Thus, we have shown that both models exhibit similar: (1) vascular Vv; (2) shifts in Vv of nonvascular components; (3) stages of neovascularization, and (4) mechanisms of neovascularization. In conclusion, coronary neovascularization occurring in oculo closely mimics normal coronary vessel development.

Normal left ventricular diastolic compliance after regression of hypertrophy.

We wished to determine whether (a) left ventricular (LV) diastolic chamber compliance and tissue elastic modulus were decreased with hypertrophy and improved after reversal of hypertension and regression of hypertrophy and whether collagen concentration was a major determinant of LV chamber compliance during hypertrophy or aging. Spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), aged 8 months at time of study, were used for the hypertension-regression experiments. The aging study was based on 14- and 24-month-old Fischer-344 rats. LV chamber compliance was measured in isolated perfused hearts arrested in diastole. Length-tension curves (tissue elastic modulus) were obtained from ventricular strips, and hydroxyproline assays were used to estimate LV collagen content. Captopril and hydrochlorothizide were given for an 8-week period to both normotensive and hypertensive rats. Treatment normalized arterial pressure and caused regression of LV hypertrophy in SHR. LV diastolic compliance was less (pressure-volume curve steeper) in SHR than in WKY, but stiffness was similar in the two groups, as indicated by similar slopes when volume was adjusted for heart mass. Treatment significantly decreased ventricular stiffness in both SHR and WKY. Length-tension curves were almost identical in SHR and WKY, but treated SHR demonstrated less tension per given length. These changes occurred although collagen did not decrease in parallel to the decrease in LV mass. Aging was associated with 66 and 60% increases in collagen content and concentration, respectively, but did not alter LV chamber compliance significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiogenesis in the uterus: potential regulation and relation to tumor angiogenesis.

Except under certain pathological conditions such as wound healing and solid tumor growth, angiogenesis is a relatively rare event in the adult. One exception, however, is the angiogenesis that occurs during the cyclical changes in the female reproductive tract. Many factors, chemical as well as mechanical, have been shown to be capable of promoting or inhibiting angiogenesis in vivo and in vitro. However, despite intense research efforts, the mechanisms involved in the regulation of angiogenesis in vivo are not fully understood. In this article we briefly review the basic steps involved in angiogenesis and present examples of factors and conditions that may serve as potential regulators of angiogenesis in the nonpregnant uterus. Finally, we discuss some of the architectural, anatomical, and physiological differences between the microcirculatory beds established during normal, self-limited vessel growth and that associated with the uncontrolled, pathological vascular growth that accompanies tumor growth and metastasis.