湖北宜昌乔家坪村寒武系水井沱组海绵动物化石群

海绵动物是最原始的动物门类之一,其演化史对于研究多细胞动物的起源和早期演化至关重要.现有的化石记录显示,水体较深的贫氧环境可能是寒武纪早期海绵动物演化的重要场所之一.前人在贵州、湖南、安徽等多地的斜坡至盆地相黑色页岩中已经报道了数个海绵化石群.在这样的环境中,由于水动力作用较弱,海绵骨架结构容易得到原位保存.湖北宜昌秭...     

Status and Perspective of Sponge Chemosystematics

In addition to their pharmaceutical applications, sponges are an important source of compounds that are used to elucidate classification patterns and phylogenetic relationships. Here we present a review and outlook on chemosystematics in sponges in seven sections: Secondary metabolites in sponges; Further applications of bioactive compound research in sponges; Sponge chemotaxonomy; Pitfalls of sponge chemotaxonomy; The chemotaxonomic suitability of sponge compounds; Potential synapomorphic markers in sponges; and The future of sponge chemotaxonomy.     

Non-rigid cryptic sponges in oyster patch reefs (Lower Kimmeridgian, Langenberg/Oker, Germany)

Summary Two patch reefs which predominately consist of the oysterNanogyra nana (Sowerby 1822) are exposed in Lower Kimmeridigian strata of the Langenberg hillrange, central Germany. Left oyster valves making up the frame-work of the reefs formed small abundant cavities that were inhabited by a unique sponge community. The excellent preservation of non-rigid sponges was related to early organomineralization within the decaying sponge tissue. As a process of sponge taphonomy, different types of microbially induced carbonates precipitated preserving spicule aggregates. Organomineralization within sponge soft tissues is especially favored with the Langenberg patch reefs due to the closed or semi-closed system conditions with the cavities. The δ¹³C values ofin situ formed microbialities reveal that carbonate precipitation was in equilibrium with Jurassic seawater. The carbon of the microbialites does not derive from the bacterial remineralization of organic matter, but is of a marine source. Likewise, organomineralization is probably related to bacterial EPS or decaying sponge tissues providing an organic matrix for initial carbonate precipitation. Biomarker analyses revealed, that the patch reef microbialites contain terminally branched fatty acids (iso-andanteiso-pentadecanoic acid) in significant concentrations. These fatty acids, like hopanoid hydrocarbons, are most likely of a bacterial source. This is in agreement with sulfate-reducing bacteria remineralizing the decaying sponges as further indicated by the occurrence of framboidal pyrite in sponge microbialites.     

Dark CO2 fixation into phospholipid-derived fatty acids by the cold-water coral associated sponge Hymedesmia (Stylopus) coriacea (Tisler Reef,NE Skagerrak)

ABSTRACT

Many cold-water sponges harbour microorganisms of which the role in the sponge host remains enigmatic. Here, we show a transfer of fixed inorganic carbon by sponge-associated microbes to its host, the cold-water coral encrusting sponge Hymedesmia (Stylopus) coriacea. Sponge were collected at approx. 100?m depth and incubated for 1.5–2.5 days with ¹³C labelled dissolved inorganic carbon (DIC) as tracer. Total DIC fixation rates ranged from 0.03–0.11?mmol C?×?mmol C_sponge × d^?1. ¹³C-tracer was recovered in bacterial-specific (i.e. short and branched) and sponge-specific (very long-chained) phospholipid-derived fatty acids (PLFA's), but was not incorporated into archaeal lipids. ¹³C-incorporation in biomarkers such as C16:1w7c and C18:1w7c indicated that nitrifying and/or sulphur-oxidizing bacteria (chemoautotrophs) were likely active in the sponge. Trophic transfer of microbially-fixed carbon to the sponge host was confirmed by recovery of label in very long chain fatty acids (VLCFA's) including C26:2 and C26:3. Tracer accumulation into several VLCFA's continued after removal of ¹³C-DIC, while tracer in most bacteria-specific PLFA's declined, indicating a transfer and elongation of bacterial-specific PLFA's to sponge-specific PLFA's. This implies that PLFA precursors released from chemo- as well as heterotrophic microbes in sponges contributed to the synthesis of VLCFA's, identifying sponge-associated bacteria as symbionts of the sponge.     

Sea-level changes as controlling factor of early diagenesis: the reefal limestones of Adnet (Late Triassic,Northern Calcareous Alps,Austria)

The uppermost Rhaetian Adnet reef is part of the Dachstein carbonate platform and is situated at the transition to the intrashelf Kössen Basin. Its diagenetic evolution is investigated focusing on dissolution cavities in the Tropfbruch quarry of Adnet (near Salzburg) stratigraphically situated immediately below the Triassic–Jurassic boundary. Sea-level changes due to global eustatic trends and regional tectonics are assumed to be the controlling factors in the development of a manifold diagenetic sequence characterized by phases of meteoric dissolution, marine and burial cementation, and internal sedimentation. Despite small-scale variations of the sequence, a superordinate pattern of diagenetic phases could be elaborated. Small-scale eustatic sea-level falls subordinate to a global regression trend caused subaerial exposures of the Adnet reef in the latest Rhaetian to earliest Hettangian. The result was karstification and meteoric dissolution of aragonitic coral skeletons (Retiophyllia) leading to the formation of biomoldic porosity. Coral septa which escaped dissolution were transformed into neomorphic calcite spar under meteoric–phreatic conditions. A first generation of dog-tooth cements precipitated sporadically on the altered coral skeletons. Eustatic sea-level rise in Early to Mid-Hettangian times caused a renewed flooding of the pore space of the Adnet reef by marine water and the influx of a first generation of internal sediments (IS I), derived from the karstified host rock of the Upper Rhaetian reef limestone. These internal sediments are overgrown by radiaxial-fibrous calcites (RFCs) whose oxygen-isotopic signature (δ¹⁸O = ?1.3 (±0.7)‰) indicates precipitation in deeper (colder) water (18–21°C) due to a first phase of drowning. An intermediate phase of eustatic sea-level lowstand in the Late Hettangian is expressed by dissolution and corrosion of RFCs. Rapid drowning of the Dachstein carbonate platform due to eustatic sea-level rise and tectonic movements took place in the Early Sinemurian and a second generation of internal sediments (IS II) derived from the Lower Sinemurian Adnet Formation is washed into the dissolution cavities. Where IS II is absent, RFCs are overgrown by a second generation of dog-tooth cements with a bright-luminescent outer rim indicating the transition to negative redox conditions in the pore water during shallow burial. Burial diagenesis is represented by blocky calcite cements which occlude the remaining pore space. Depleted oxygen-isotope values and significant Fe contents indicate precipitation under reducing redox conditions and elevated temperatures of 30–50°C at burial depths of 420–870 m. Locally, replacive saddle dolomite is the latest diagenetic phase in the Adnet reef indicating crystallization under hydrothermal influences related to compressional subduction regimes of the Penninic Ocean.     

Discovery of a “Living Dinosaur”: Globally unique modern hexactinellid sponge reefs off British Columbia, Canada

Summary Globally unique hexactinellid sponge reefs occur on the continental shelf off British Columbia, Canada. They cover about 425 km² of seafloor on the continental shelf off British Columbia (Canada) in water depths between 165 and 240 metres and occur on a low-angle deep shelf, iceberg scoured seafloor, characterized by very low sedimentation rates and very stable environmental conditions. The sponge bioherms are up to 19 metres high with steep flanks, whereas the biostromes are 2–10 metres thick and many kilometres wide. They all consist of dense populations of only seven hexactinellid species. Three of them, all hexactinosan species (Aphrocallistes vastus, Heterochone calyx, Farrea occa) are the main frambuilders, composing a true rigid framework of sponge skeletons encased in a organic rich matrix of modern clay baffled by the sponges. Growth rates of hexactinosan sponges range in the order of 0–7 centimetres per year. The base of the oldest sponge reefs date from approximately 9000 years b.p. Different invertebrate and fish faunas occupy the reefs than occur on adjacent seafloor areas and some species appear to use the sponge reef complex structures as refugia where they can hide. Sidescan sonar data and direct observation by manned submersible clearly show that large areas of sponge reefs have been heavily damaged by seafloor trawling in the past decade. These unique extant siliceous sponge reefs can be used as a modern analogue for a better understanding and interpretation of fossil siliceous sponge reefs, known from many ages and many locations world wide.     

Efforts to develop a cultured sponge cell line: revisiting an intractable problem

Residents of the marine environment, sponges (Porifera) have the ability to produce organic compounds known as secondary metabolites, which are not directly involved in the normal growth, development, or reproduction of an organism. Because of their sessile nature, the production of these bioactive compounds has been interpreted as a functional adaptation to serve in an important survival role as a means to counter various environmental stress factors such as predation, overgrowth by fouling organisms, or competition for limited space. Regardless of the reasons for this adaptation, a variety of isolated compounds have already proven to demonstrate remarkable anticancer, fungicidal, and antibiotic properties. A major obstacle to the isolation and production of novel compounds from sponges is the lack of a large, reliable source of sponge material. Sponge collection from the sea would be environmentally detrimental to the already stressed and sparse sponge populations. Sponge production in an aquaculture setting might appear to be an ideal alternative but would also be cost-ineffective and sponge growth is extremely slow. A third approach involves the development of a sponge cell culture system capable of producing the necessary cell numbers to harvest for research purposes as well as for the eventual commercial-scale production of promising bioactive compounds. Unfortunately, little progress has been made in this direction other than the establishment of temporary cultures containing aggregates and fragments of cells. One impediment toward successful sponge cell culture might be ascribed to a lack of published knowledge of failed methodologies, and thus, time and effort is wasted on continued reinvention of the same methods and procedures. Consequently, we have undertaken here to chart some of our unsuccessful research efforts, our methodology, and results to provide the sponge research community with knowledge to assist them to better avoid taking the same failed pathways.     

贵州寒武纪海绵动物化石组合特征

贵州寒武纪海绵化石丰富。分布广泛,主要产于早寒武世梅树村期的戈仲伍组或牛蹄塘组底部、筇竹寺期的牛蹄塘组和早、中寒武世凯里组。梅树村期的海绵属种单调,主要为六射海绵类麦粒海绵（Triticispongia sp．）及一些海绵骨针,代表寒武纪海绵首次爆发或辐射的前奏和先驱。牛蹄塘组海绵多达13个属,是寒武纪海绵辐射的重要窗口,以普通海绵略占优势,Leptomitus为优势属。凯里组的海绵共计7属,以普通海绵为主。贵州寒武纪海绵动物群的发展具有明显的阶段性和组成的差异性,其演变过程受到环境的制约。贵州早期海绵动物的发现和研究不仅改变了寒武纪生物多样性的认识,而且还为探讨海绵动物本身的起源、辐射提供了重要材料。     

Kieselschwämme aus dem unterjurassischen Misonekalk der Trento-Plattform (Südalpen): Taxonomie und phylogenetische Relevanz

The Lower Jurassic Misone Limestone of the Trento Platform (Southern Alps, Italy) contains a siliceous sponge fauna which is here described. Besides the well-known Moroccan sponge carbonates, these Lower Jurassic spongioliths from the Trento Platform are presently the second mass occurrence of siliceous sponges, which is known from the southern margin of the Tethys. They differ from each other in regard of the composition of the sponge fauna and the absence of microbial crusts in the spongioliths of the Trento Platform. There, hexactinosans and lithistid demosponges occur in equal proportions. Sphinctozoans are another very characteristic element. Because of the richness in both sphinctozoans and siliceous sponges, the Trento occurrences may be considered as a transitional fauna between the late Palaeozoic-Triassic sponge fauna dominated by sphinctozoans and the post-Liassic sponge fauna dominated by more modern groups of siliceous sponges. Two new siliceous sponge genera with their species are established:Misonia baldensis n. gen. n. sp. (Hexactinosa) andBenacia princeps n. gen. n. sp. (lithistid Demospongiae). The rarity of siliceous sponge dominated spongioliths in the Early Jurassic is due to the restricted occurrence of low energy, deeper shelf areas.     

Sponge Carrying by Dolphins (Delphinidae,Tursiops sp.): A Foraging Specialization Involving Tool Use?

During long-term research on bottlenose dolphins (Tursiops sp.) in Shark Bay, Western Australia, several individuals were observed carrying sponges, Echinodictyum mesenterinum, on their rostra. Over multiple years, five regularly sighted individuals were usually carrying sponges when encountered (67–100% of encounters). Four additional regularly sighted individuals were observed with sponges just one time each. All five individuals that routinely carried sponges were female. Two of the anomalous, one-time carriers were female, one was likely female, and one was male. Most observations of sponge carrying occurred within a restricted area, a relatively deep water channel (8–10 m deep). Surface observations of sponge carrying, including focal animal observations, revealed a stereotyped surfacing and diving pattern, and occasional indications of prey consumption. Three hypotheses are considered regarding the function of sponge carrying: 1. dolphins were playing with the sponges; 2. the sponges contain some compound of use to the dolphins (e.g. for medicinal purposes); and 3. the sponges were used as a tool to aid in foraging. The foraging tool hypothesis is best supported, but the exact manner in which sponges are used remains to be discovered. Sponge carrying is a behavioural specialization, probably involving foraging, and regularly engaged in by only a small proportion of female dolphins in Shark Bay.     

Nitrogen Biogeochemistry in the Caribbean Sponge,Xestospongia muta: A Source or Sink of Dissolved Inorganic Nitrogen?

Background

Sponges have long been known to be ecologically important members of the benthic fauna on coral reefs. Recently, it has been shown that sponges are also important contributors to the nitrogen biogeochemistry of coral reefs. The studies that have been done show that most sponges are net sources of dissolved inorganic nitrogen (DIN; NH₄ ⁺ and NO₃ ⁻) and that nitrification, mediated by their symbiotic prokaryotes, is the primary process involved in supplying DIN to adjacent reefs.

Methodology/Principal Findings

A natural experiment was conducted with the Caribbean sponge Xestospongia muta from three different locations (Florida Keys, USA; Lee Stocking Island, Bahamas and Little Cayman, Cayman Islands). The DIN fluxes of sponges were studied using nutrient analysis, stable isotope ratios, and isotope tracer experiments. Results showed that the fluxes of DIN were variable between locations and that X. muta can be either a source or sink of DIN. Stable isotope values of sponge and symbiotic bacterial fractions indicate that the prokaryotic community is capable of taking up both NH₄ ⁺ and NO₃ ⁻ while the differences in δ ¹⁵N between the sponge and bacterial fractions from the NH₄ ⁺ tracer experiment suggest that there is translocation of labeled N from the symbiotic bacteria to the host.

Conclusions/Significance

Nitrogen cycling in X. muta appears to be more complex than previous studies have shown and our results suggest that anaerobic processes such as denitrification or anammox occur in these sponges in addition to aerobic nitrification. Furthermore, the metabolism of this sponge and its prokaryotic symbionts may have a significant impact on the nitrogen biogeochemistry on Caribbean coral reefs by releasing large amounts of DIN, including higher NH₄ ⁺ concentrations that previously reported.     

The Homoscleromorph sponge Oscarella lobularis,a promising sponge model in evolutionary and developmental biology

Sponges branch basally in the metazoan phylogenetic tree and are believed to be composed of four distinct lineages with still uncertain relationships. Indeed, some molecular studies propose that Homoscleromorpha may be a fourth Sponge lineage, distinct from Demospongiae in which they were traditionally classified. They harbour many features that distinguish them from other sponges and are more evocative of those of the eumetazoans. They are notably the only sponges to possess a basement membrane with collagen IV and specialized cell‐junctions, thus possessing true epithelia. Among Homoscleromorphs, we have chosen Oscarella lobularis as a model species. This common and easily accessible sponge is characterized by relatively simple histology and cell composition, absence of skeleton, and strongly pronounced epithelial structure. In this review, we explore the specific features that make O. lobularis a promising homoscleromorph sponge model for evolutionary and developmental researches.     

Facies of Liassic sponge mounds, central High Atlas, Morocco

Summary Liassic sponge mounds of the central High Atlas (Rich area, northern Morocco) have a stratigraphic range from the Lower/Upper Sinemurian boundary interval up to the lower parts of the Lower Pliensbachian (Carixian). The base of Liassic sponge mounds consists of a transgressive discontinuity, i.e., a condensed section of microbioclastic wackestones with firm- and hardgrounds, ferruginous stromatolites, sponge spicules and ammonites. The top of Liassic sponge mounds is an irregular palaeorelief covered by cherty marl-limestone rhythmites, namely hemipelagic spicular wackestones with radiolaria. In the Rich area, section Foum Tillicht, the sponge mound succession has a total thickness of about 250 meters. Within this succession we distinguished between three mound intervals. The lower mound interval shows only small, meter-scale sponge mounds consisting of boundstones with lyssakine sponges, commensalicTerebella and the problematicumRadiomura. This interval forms a shallowing-upward sequence culminating in a bedded facies withTubiphytes, calcareous algae (Palaeodasycladus), sponge lithoclasts, coated grains, and thin rims of marine cement. The middle mound interval is aggradational with decametric mounds and distinct thrombolitic textures and reefal cavities. The mound assemblage here consists of hexactinellid sponges, lithistid demosponges, non-rigid demosponges,Radiomura, Serpula (Dorsoserpula), Terebella, encrusting bryozoa, and minor contributions by calcareous sponges, and excavating sponges (typeAka). Thrombolites are dendrolitic and may reach sizes of several tens of centimeters, similar to the maximum size of siliceous sponges. The upper mound interval appears retrogradational and geometries change upsection from mound shapes to flat lenses and level-bottom, biostromal sponge banks. The biotic assemblage is similar to that of the middle mound interval and there is no difference between mound and bank communities. The demise of sponge mounds is successive from regional spread in the Sinemurian to more localised spots in the Lower Pliensbachian. This reduction correlates with an increasing influence of pelagic conditions. At Foum Tillicht, sponge mounds lack any photic contribution and there is virtually no differentiation into subcommunities between mound surface and cavity dwelling organisms. There is some evidence that the heterotrophic food web of mound communities was sourced by oxygen minimum zone edge effects, namely microbial recycling of essential elements such as N and P. Basin geometry suggests a waterdepth of several 100's of meters, well below the photic zone and possibly only controlled by the depth range of the oxygen minimum zone. Palaeoceanographic conditions of well-stratified deeper water masses diminished gradually during widespread transgression across the Sinemurian to Pliensbachian boundary culminating in the Lower Pliensbachianibex ammonite zone.     

Sponge assemblages of the deep Weddell Sea: ecological and zoogeographic results of ANDEEP I–III and SYSTCO I expeditions

The aim of this study is to analyze the community structure, ecology and distribution of deep Weddell Sea sponge faunas. Analysis was performed on the basis of sponges sampled during ANDEEP I–III and SYSTCO I expeditions (2002–2008) by RV Polarstern. The material obtained comprises about 800 sponge specimens, representing 129 species, within these are 95 species of demosponges (including 15 new to science), 25 hexactinellid species (7 new) and 9 calcarean species (5 new). Sponges were sampled at 51 stations in depths between 500 and 5,500 m. At most stations, sponge densities were very low, and many species are represented by one or two specimens only. Community structure by Bray–Curtis similarity was analyzed as well as depth range and spatial distribution of the most common species. Zoogeographic affinities of sampled faunas are analyzed. Three associations of sponges are found in the deep Weddell Sea: (1) The Polymastia/Tentorium community, (including Rossella associations) distributed on the lower shelf and continental slope. (2) The Bathydorus community, distributed on the continental slope and upper abyssal. (3) The Caulophacus community, associated with Cladorhizidae, is characteristic for the abyssal plains. The associations follow each other successively both bathymetrically and geographically, from shallow to deep, from shelf and ridge structures into the open abyssal. A distinct faunistic boundary between shelf and deep sea is not present. In general, the sponge fauna of the deep Weddell Sea is regionally restricted and shows stronger affinities only to the sponge fauna of the subantarctic islands.     

Barcoding sponges: an overview based on comprehensive sampling

Background

Phylum Porifera includes ∼8,500 valid species distributed world-wide in aquatic ecosystems ranging from ephemeral fresh-water bodies to coastal environments and the deep-sea. The taxonomy and systematics of sponges is complicated, and morphological identification can be both time consuming and erroneous due to phenotypic convergence and secondary losses, etc. DNA barcoding can provide sponge biologists with a simple and rapid method for the identification of samples of unknown taxonomic membership. The Sponge Barcoding Project (www.spongebarcoding.org), the first initiative to barcode a non-bilaterian metazoan phylum, aims to provide a comprehensive DNA barcode database for Phylum Porifera.

Methodology/Principal Findings

∼7,400 sponge specimens have been extracted, and amplification of the standard COI barcoding fragment has been attempted for approximately 3,300 museum samples with ∼25% mean amplification success. Based on this comprehensive sampling, we present the first report on the workflow and progress of the sponge barcoding project, and discuss some common pitfalls inherent to the barcoding of sponges.

Conclusion

A DNA-barcoding workflow capable of processing potentially large sponge collections has been developed and is routinely used for the Sponge Barcoding Project with success. Sponge specific problems such as the frequent co-amplification of non-target organisms have been detected and potential solutions are currently under development. The initial success of this innovative project have already demonstrated considerable refinement of sponge systematics, evaluating morphometric character importance, geographic phenotypic variability, and the utility of the standard barcoding fragment for Porifera (despite its conserved evolution within this basal metazoan phylum).     

Predictors of the distribution and abundance of a tube sponge and its resident goby

Microhabitat specialists offer tractable systems for studying the role of habitat in determining species’ distribution and abundance patterns. While factors underlying the distribution patterns of these specialists have been studied for decades, few papers have considered factors influencing both the microhabitat and the inhabitant. On the Belizean barrier reef, the obligate sponge-dwelling goby Elacatinus lori inhabits the yellow tube sponge Aplysina fistularis. We used field data and multivariate analyses to simultaneously consider factors influencing sponge and goby distributions. Sponges were non-randomly distributed across the reef with density peaking at a depth of 10–20 m. Sponge morphology also varied with depth: sponges tended to be larger and have fewer tubes with increasing depth. Knowing these patterns of sponge distribution and morphology, we considered how they influenced the distribution of two categories of gobies: residents (≥18 mm SL) and settlers (<18 mm SL). Maximum tube length, number of sponge tubes, and depth were significant predictors of resident distribution. Residents were most abundant in large sponges with multiple tubes, and were virtually absent from sponges shallower than 10 m. Similarly, maximum tube length and number of sponge tubes were significant predictors of settler distribution, with settlers most abundant in large sponges with multiple tubes. The presence or absence of residents in a sponge was not a significant predictor of settler distribution. These results provide us with a clear understanding of where sponges and gobies are found on the reef and support the hypothesis that microhabitat characteristics are good predictors of fish abundance for species that are tightly linked to microhabitat.     

Identifying miRNA sponge modules using biclustering and regulatory scores

Background

MicroRNA (miRNA) sponges with multiple tandem miRNA binding sequences can sequester miRNAs from their endogenous target mRNAs. Therefore, miRNA sponge acting as a decoy is extremely important for long-term loss-of-function studies both in vivo and in silico. Recently, a growing number of in silico methods have been used as an effective technique to generate hypotheses for in vivo methods for studying the biological functions and regulatory mechanisms of miRNA sponges. However, most existing in silico methods only focus on studying miRNA sponge interactions or networks in cancer, the module-level properties of miRNA sponges in cancer is still largely unknown.

Results

We propose a novel in silico method, called miRSM (miRNA Sponge Module) to infer miRNA sponge modules in breast cancer. We apply miRSM to the breast invasive carcinoma (BRCA) dataset provided by The Cancer Genome Altas (TCGA), and make functional validation of the computational results. We discover that most miRNA sponge interactions are module-conserved across two modules, and a minority of miRNA sponge interactions are module-specific, existing only in a single module. Through functional annotation and differential expression analysis, we also find that the modules discovered using miRSM are functional miRNA sponge modules associated with BRCA. Moreover, the module-specific miRNA sponge interactions among miRNA sponge modules may be involved in the progression and development of BRCA. Our experimental results show that miRSM is comparable to the benchmark methods in recovering experimentally confirmed miRNA sponge interactions, and miRSM outperforms the benchmark methods in identifying interactions that are related to breast cancer.

Conclusions

Altogether, the functional validation results demonstrate that miRSM is a promising method to identify miRNA sponge modules and interactions, and may provide new insights for understanding the roles of miRNA sponges in cancer progression and development.

     

Innervation and neurokinin receptors during angiogenesis in the rat sponge granuloma

Summary Angiogenesis is an essential component of wound healing and inflammation. In the rat subcutaneous sponge implantation model, angiogenesis can be enhanced by administration of the sensory neuropeptide, substance P. We have used quantitativein vitro receptor autoradiography and immunohistochemistry to investigate the development of endogenous neurovascular regulatory systems in the newly-formed granulation tissue of this model. The fraction of endothelial cells immunoreactive for proliferating cell nuclear antigen, endothelial fractional area, and¹³³Xe clearance were used as measures of endothelial proliferation, neovascularization, and blood flow, respectively. Endothelial proliferation occurred predominantly in tissues surrounding the sponge, and peaked before neovascularization of sponge stroma and the establishment of sponge blood flow. Substance P-containing sensory nerves and specific, high affinity substance P binding sites with characteristics of neurokinin receptors of the NK₁ subclass, were localized to microvessels surrounding the sponge at all time points. Lower density substance P binding sites were localized to newly formed microvessels within the sponge stroma, progressively increasing in density from day 4 to day 14. Nerve fibres were observed in the stroma of only 2 of 6 sponges at day 14, and none at earlier time points. These data support the hypothesis that substance P-enhanced angiogenesis in this model results from a direct action on microvascular NK₁ receptors. Neovascularization is a sequential process, with early endothelial proliferation followed by new vessel formation and increased blood flow, with maturation of endogenous neurovascular regulatory systems occurring late in this process in inflamed tissues.