Rationale Inactivating mutations in the FOXF1 gene locus are frequently found in patients with Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACD/MPV), a lethal congenital disorder, which is characterized by severe abnormalities in the respiratory, cardio-vascular and gastro-intestinal systems. In mice, haploinsufficiency of the Foxf1 gene causes alveolar capillary dysplasia and developmental defects in lung, intestinal and gall bladder morphogenesis. Objective While FOXF1 is expressed in multiple mesenchyme-derived cell types, cellular origins and molecular mechanisms of developmental abnormalities in FOXF1-deficient mice and ACD/MPV patients remain uncharacterized due to lack of mouse models with cell-restricted inactivation of the Foxf1 gene. In the present study, the role of FOXF1 in endothelial cells was examined using a conditional knockout approach. Methods and Results A novel mouse line harboring Foxf1-floxed alleles was generated by homologous recombination. Tie2-Cre and Pdgfb-CreER transgenes were used to delete Foxf1 from endothelial cells. FOXF1-deficient embryos exhibited embryonic lethality, growth retardation, polyhydramnios, cardiac ventricular hypoplasia and vascular abnormalities in the lung, placenta, yolk sac and retina. Deletion of FOXF1 from endothelial cells reduced endothelial proliferation, increased apoptosis, inhibited VEGF signaling and decreased expression of endothelial genes critical for vascular development, including VEGF receptors Flt1 and Flk1, Pdgfb, Pecam1, CD34, integrin β3, ephrin B2, Tie2 and the non-coding RNA Fendrr. ChIP assay demonstrated that Flt1, Flk1, Pdgfb, Pecam1 and Tie2 genes are direct transcriptional targets of FOXF1. Conclusions FOXF1 is required for formation of embryonic vasculature by regulating endothelial genes critical for vascular development and VEGF signaling.

BackgroundThe nerve net of Nematostella is generated using a conserved cascade of neurogenic transcription factors. For example, NvashA, a homolog of the achaete-scute family of basic helix-loop-helix transcription factors, is necessary and sufficient to specify a subset of embryonic neurons. However, positive regulators required for the expression of neurogenic transcription factors remain poorly understood.ResultsWe show that treatment with the MEK/MAPK inhibitor U0126 severely reduces the expression of known neurogenic genes, Nvath-like, NvsoxB(2), and NvashA, and known markers of differentiated neurons, suggesting that MAPK signaling is necessary for neural development. Interestingly, ectopic NvashA fails to rescue the expression of neural markers in U0126-treated animals. Double fluorescence in situ hybridization and transgenic analysis confirmed that NvashA targets represent both unique and overlapping populations of neurons. Finally, we used a genome-wide microarray to identify additional patterning genes downstream of MAPK that might contribute to neurogenesis. We identified 18 likely neural transcription factors, and surprisingly identified ~40 signaling genes and transcription factors that are expressed in either the aboral domain or animal pole that gives rise to the endomesoderm at late blastula stages.ConclusionsTogether, our data suggest that MAPK is a key early regulator of neurogenesis, and that it is likely required at multiple steps. Initially, MAPK promotes neurogenesis by positively regulating expression of NvsoxB(2), Nvath-like, and NvashA. However, we also found that MAPK is necessary for the activity of the neurogenic transcription factor NvashA. Our forward molecular approach provided insight about the mechanisms of embryonic neurogenesis. For instance, NvashA suppression of Nvath-like suggests that inhibition of progenitor identity is an active process in newly born neurons, and we show that downstream targets of NvashA reflect multiple neural subtypes rather than a uniform neural fate. Lastly, analysis of the MAPK targets in the early embryo suggests that MAPK signaling is critical not only to neurogenesis, but also endomesoderm formation and aboral patterning.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0282-1) contains supplementary material, which is available to authorized users.

The organization of cnidarian nerve nets is traditionally described as diffuse with randomly arranged neurites that show minimal reproducibility between animals. However, most observations of nerve nets are conducted using cross-reactive antibodies that broadly label neurons, which potentially masks stereotyped patterns produced by individual neuronal subtypes. Additionally, many cnidarians species have overt structures such as a nerve ring, suggesting higher levels of organization and stereotypy exist, but mechanisms that generated that stereotypy are unknown. We previously demonstrated that NvLWamide-like is expressed in a small subset of the Nematostella nerve net and speculated that observing a few neurons within the developing nerve net would provide a better indication of potential stereotypy. Here we document NvLWamide-like expression more systematically. NvLWamide-like is initially expressed in the typical neurogenic salt and pepper pattern within the ectoderm at the gastrula stage, and expression expands to include endodermal salt and pepper expression at the planula larval stage. Expression persists in both ectoderm and endoderm in adults. We characterized our NvLWamide-like::mCherry transgenic reporter line to visualize neural architecture and found that NvLWamide-like is expressed in six neural subtypes identifiable by neural morphology and location. Upon completing development the numbers of neurons in each neural subtype are minimally variable between animals and the projection patterns of each subtype are consistent. Furthermore, between the juvenile polyp and adult stages the number of neurons for each subtype increases. We conclude that development of the Nematostella nerve net is stereotyped between individuals. Our data also imply that one aspect of generating adult cnidarian nervous systems is to modify the basic structural architecture generated in the juvenile by increasing neural number proportionally with size.

Background The ability to regenerate body parts is a feature of metazoan organisms and the focus of intense research aiming to understand its basis. A number of mechanisms involved in regeneration, such as proliferation and tissue remodeling, affect whole tissues; however, little is known on how distinctively different constituent cell types respond to the dynamics of regenerating tissues. Preliminary studies suggest that a number of organisms alter neuronal numbers to scale with changes in body size. In some species with the ability of whole-body axis regeneration, it has additionally been observed that regenerates are smaller than their pre-amputated parent, but maintain the correct morphological proportionality, suggesting that scaling of tissue and neuronal numbers also occurs. However, the cell dynamics and responses of neuronal subtypes during nervous system regeneration, scaling, and whole-body axis regeneration are not well understood in any system. The cnidarian sea anemone Nematostella vectensis is capable of whole-body axis regeneration, with a number of observations suggesting the ability to alter its size in response to changes in feeding. We took advantage of Nematostella’s transparent and “simple” body plan and the NvLWamide-like mCherry fluorescent reporter transgenic line to probe the response of neuron populations to variations in body size in vivo in adult animals during body scaling and regeneration. Results We utilized the previously characterized NvLWamide-like::mCherry transgenic reporter line to determine the in vivo response of neuronal subtypes during growth, degrowth, and regeneration. Nematostella alters its size in response to caloric intake, and the nervous system responds by altering neuronal number to scale as the animal changes in size. Neuronal numbers in both the endodermal and ectodermal nerve nets decreased as animals shrunk, increased as they grew, and these changes were reversible. Whole-body axis regeneration resulted in regenerates that were smaller than their pre-amputated size, and the regenerated nerve nets were reduced in neuronal number. Different neuronal subtypes had distinct responses during regeneration, including consistent, not consistent, and conditional increases in number. Conditional responses were regulated, in part, by the size of the remnant fragment and the position of the amputation site. Regenerates and adults with reduced nerve nets displayed normal behaviors, indicating that the nerve net retains functionality as it scales. Conclusion These data suggest that the Nematostella nerve net is dynamic, capable of scaling with changes in body size, and that neuronal subtypes display differential regenerative responses, which we propose may be linked to the scale state of the regenerating animals.

Females of many spider species invest in chemical advertisements to attract males, yet variation in investment relative to the presence or quality of males remains poorly understood. Males of the wolf spider Pardosa milvina court females longer and more intensively when in contact with female silk and also court more intensively when encountering silk from virgin rather than mated females; therefore, females may use silk as a medium to advertise their receptivity to mate. We estimated female investment in advertisements by measuring variation in the quantity and type of silk deposited by females in the presence or absence of males and among males that varied in their courtship intensity. We measured dragline silk, cord silk, and attachment disk deposition from females on gridded sheets of paper in response to four stimuli over a 30-min period (n = 39/treatment): (1) an intensively (high) courting male with access to female silk, (2) a weakly courting or non-courting male (low) without access to female silk, (3) no male present, but female silk present (silk control), and (4) no stimulus present (control). Females produced significantly more dragline silk and significantly less cord silk in the presence of low-courting males compared to any other treatment, but we found no difference in attachment disk deposition across treatments. Our results suggest that females invest more heavily in dragline deposition when encountering low-courting males. Additional studies are necessary to determine the relative role of different silk types in male-female sexual communication.