Reduction of embryonic E93 expression as a hypothetical driver of the evolution of insect metamorphosis.

The early embryo of the cockroach Blattella germanica exhibits high E93 expression. In general, E93 triggers adult morphogenesis during postembryonic development. Here we show that E93 is also crucial in early embryogenesis in the cockroach, as a significant number of E93-depleted embryos are unable to develop the germ band under maternal RNAi treatment targeting E93. Moreover, transcriptomic analysis indicates that E93 depletion results in important gene expression changes in the early embryo, and many of the differentially expressed genes are involved in development. Then, using public databases, we gathered E93 expression data in embryo and preadult stages, finding that embryonic expression of E93 is high in hemimetabolan species (whose juveniles, or nymphs, are similar to the adult) and low in holometabolans (whose juveniles, or larvae, are different from the adult). E93 expression is also low in Thysanoptera and in Hemiptera Sternorrhyncha, hemimetabolans with postembryonic quiescent stages, as well as in Odonata, the nymph of which is very different from the adult. In ametabolans, such as the Zygentoma Thermobia domestica, E93 transcript levels are very high in the early embryo, whereas during postembryonic development they are medium and relatively constant. We propose the hypothesis that during evolution, a reduction of E93 expression in the embryo of hemimetabolans facilitated the larval development and the emergence of holometaboly. Independent decreases of E93 transcripts in the embryo of Odonata, Thysanoptera, and different groups of Hemiptera Sternorrhyncha would have allowed the development of modified juvenile stages adapted to specific ecophysiological conditions.

Broad complex and wing development in cockroaches.

In hemimetabolan insects, the transcription factor Broad complex (Br-C) promotes wing growth and development during the nymphal period. We wondered whether Br-C could trigger the initiation of wing development, using the cockroach Blattella germanica as a model. We show that first instar nymphs have their unique identity of these three thoracic segments specified. During embryogenesis, the expression of Br-C and some wing-related genes show two matching waves. The first takes place before the formation of the germ band, which might be involved in the establishment of various developmental fields including a potential "wing field", and the second wave around organogenesis, possibly involved in the initiation of wing development. However, the expression of Br-C in early embryogenesis concentrates in the developing central nervous system, thus not co-localizing with the expression of the typical wing-related gene vestigial, which is expressed at the edge of the thoracic and abdominal segments. This suggests that Br-C is not specifically involved in the establishment of a potential "wing field" in early embryogenesis. Moreover, maternal RNAi for Br-C depletes the first wave of Br-C expression but does not affect the early expression of wing-related genes. As maternal Br-C RNAi did not deplete the second expression wave of Br-C, we could not evaluate if Br-C is involved in the initiation of wing development. Alternatively, using nymphal RNAi of Br-C and Sex combs reduced (Scr), we show that Br-C contributes to the formation of ectopic wing structures that develop in the prothorax when Scr is depleted. The gene most clearly influenced by Br-C RNAi is nubbin (nub), which, in nymphs is crucial for wing growth. Together, these results suggest that Br-C does not specifically contribute to the establishment of the "wing field", but it does seem important later, in the initiation of wing development, enhancing the expression of wing-related genes, especially nub. This supports the hypothesis previously proposed by the authors, whereby Br-C might have facilitated the evolution of holometaboly. However, there is no doubt that other factors have also contributed to this evolution.

Vasa nucleates asymmetric translation along the mitotic spindle during unequal cell divisions.

mRNA translation on the spindle is hypothesized to be an essential strategy for the localized production of cell regulators. This mechanism may be important particularly in early embryonic cells, which have a large diffusion volume and that undergo rapid cell divisions. Evidence to test such a hypothesis has been, however, limited. Here, we use an embryo with both symmetric and asymmetric cell divisions and manipulate Vasa protein, an RNA-helicase, on the spindle in live sea urchin embryos. We learned that the spindle serves as a major site of translation and that protein synthesis within a single spindle can be unequal and help drive asymmetric cell divisions during embryogenesis. Recruiting Vasa to the ectopic sub-cellular region induced a new site of translation, disturbed asymmetric translation on the spindle, and changed the cell fate. Based on these observations, we conclude that Vasa functions in localized translation, which provides a spatiotemporal control in protein synthesis and is essential for rapidly developing embryonic cells.

α7 Nicotinic acetylcholine receptor mediates right ventricular fibrosis and diastolic dysfunction in pulmonary hypertension.

Right ventricular (RV) fibrosis is a key feature of maladaptive RV hypertrophy and dysfunction and is associated with poor outcomes in pulmonary hypertension (PH). However, mechanisms and therapeutic strategies to mitigate RV fibrosis remain unrealized. Previously, we identified that cardiac fibroblast α7 nicotinic acetylcholine receptor (α7 nAChR) drives smoking-induced RV fibrosis. Here, we sought to define the role of α7 nAChR in RV dysfunction and fibrosis in the settings of RV pressure overload as seen in PH. We show that RV tissue from PH patients has increased collagen content and ACh expression. Using an experimental rat model of PH, we demonstrate that RV fibrosis and dysfunction are associated with increases in ACh and α7 nAChR expression in the RV but not in the left ventricle (LV). In vitro studies show that α7 nAChR activation leads to an increase in adult ventricular fibroblast proliferation and collagen content mediated by a Ca2+/epidermal growth factor receptor (EGFR) signaling mechanism. Pharmacological antagonism of nAChR decreases RV collagen content and improves RV function in the PH model. Furthermore, mice lacking α7 nAChR exhibit improved RV diastolic function and have lower RV collagen content in response to persistently increased RV afterload, compared with WT controls. These finding indicate that enhanced α7 nAChR signaling is an important mechanism underlying RV fibrosis and dysfunction, and targeted inhibition of α7 nAChR is a potentially novel therapeutic strategy in the setting of increased RV afterload.

Tergal and pleural wing-related tissues in the German cockroach and their implication to the evolutionary origin of insect wings.

The acquisition of wings has facilitated the massive evolutionary success of pterygotes (winged insects), which now make up nearly three-quarters of described metazoans. However, our understanding of how this crucial structure has evolved remains quite elusive. Historically, two ideas have dominated in the wing origin debate, one placing the origin in the dorsal body wall (tergum) and the other in the lateral pleural plates and the branching structures associated with these plates. Through studying wing-related tissues in the wingless segments (such as wing serial homologs) of the beetle, Tribolium castaneum, we obtained several crucial pieces of evidence that support a third idea, the dual origin hypothesis, which proposes that wings evolved from a combination of tergal and pleural tissues. Here, we extended our analysis outside of the beetle lineage and sought to identify wing-related tissues from the wingless segments of the cockroach, Blattella germanica. Through detailed functional and expression analyses for a critical wing gene, vestigial (vg), along with re-evaluating the homeotic transformation of a wingless segment induced by an improved RNA interference protocol, we demonstrate that B. germanica possesses two distinct tissues in their wingless segments, one with tergal and one with pleural nature, that might be evolutionarily related to wings. This outcome appears to parallel the reports from other insects, which may further support a dual origin of insect wings. However, we also identified a vg-independent tissue that contributes to wing formation upon homeotic transformation, as well as vg-dependent tissues that do not appear to participate in wing formation, in B. germanica, indicating a more complex evolutionary history of the tissues that contributed to the emergence of insect wings.

Treatment of Pulmonary Hypertension With Angiotensin II Receptor Blocker and Neprilysin Inhibitor Sacubitril/Valsartan.

BACKGROUND: Angiotensin II has been implicated in maladaptive right ventricular (RV) hypertrophy and fibrosis associated with pulmonary hypertension (PH). Natriuretic peptides decrease RV afterload by promoting pulmonary vasodilation and inhibiting vascular remodeling but are degraded by neprilysin. We hypothesized that angiotensin receptor blocker and neprilysin inhibitor, sacubitril/valsartan (Sac/Val, LCZ696), will attenuate PH and improve RV function by targeting both pulmonary vascular and RV remodeling. METHODS: PH was induced in rats using the SU5416/hypoxia model (Su/Hx), followed by 6-week treatment with placebo, Sac/Val, or Val alone. There were 4 groups: CON-normoxic animals with placebo (n=18); PH-Su/Hx rats+placebo (n=34); PH+Sac/Val (N=24); and PH+Val (n=16). RESULTS: In animals with PH, treatment with Sac/Val but not Val resulted in significant reduction in RV pressure (mm Hg: PH: 62±4, PH+Sac/Val: 46±5), hypertrophy (RV/LV+S: PH: 0.74±0.06, PH+Sac/Val: 0.46±0.06), collagen content (µg/50 µg protein: PH: 8.2±0.3, PH+Sac/Val: 6.4±0.4), pressures and improvement in RVs (mm/s: PH: 31.2±1.8, PH+Sac/Val: 43.1±3.6) compared with placebo. This was associated with reduced pulmonary vascular wall thickness, increased lung levels of ANP (atrial natriuretic peptide), BNP (brain-type natriuretic peptide), and cGMP, and decreased plasma endothelin-1 compared with PH alone. Also, PH+Sac/Val animals had altered expression of PKC isozymes in RV tissue compared with PH alone. CONCLUSIONS: Sac/Val reduces pulmonary pressures, vascular remodeling, as well as RV hypertrophy in a rat model of PH and may be appropriate for treatment of pulmonary hypertension and RV dysfunction.

A tumor suppressor Retinoblastoma1 is essential for embryonic development in the sea urchin.

BACKGROUND: Embryonic cells and cancer cells share various cellular characteristics important for their functions. It has been thus proposed that similar mechanisms of regulation may be present in these otherwise disparate cell types. RESULTS: To explore how regulative embryonic cells are fundamentally different from cancerous cells, we report here that a fine balance of a tumor suppressor protein Retinoblastoma1 (Rb1) and a germline factor Vasa are important for proper cell proliferation and differentiation of the somatic cells during embryogenesis of the sea urchin. Rb1 knockdown blocked embryonic development and induced Vasa accumulation in the entire embryo, while its overexpression resulted in a smaller-sized embryo with differentiated body structures. These results suggest that a titrated level of Rb1 protein may be essential for a proper balance of cell proliferation and differentiation during development. Vasa knockdown or overexpression, on the other hand, reduced or increased Rb1 protein expression, respectively. CONCLUSIONS: Taken together, it appears that Vasa protein positively regulates Rb1 protein while Rb1 protein negatively regulates Vasa protein, balancing the act of these two antagonistic molecules in somatic cells. This mechanism may provide a fine control of cell proliferation and differentiation, which is essential for regulative embryonic development.

Juvenile hormone signaling in short germ-band hemimetabolan embryos.

The role of juvenile hormone (JH) in insect embryos is far from understood, especially in short germ-band hemimetabolan species. To shed light on this issue, we depleted the mRNA levels of Krüppel homolog 1, Methoprene-tolerant and JH acid O-methyltransferase, key elements of JH signaling, in embryos of the short germ-band hemimetabolan species Blattella germanica This precluded the formation of the germ-band anlage in a group of embryos. Hatchability was also reduced, which might have been caused by premature upregulation of laccase 2, a promoter of cuticle tanning. In other cases, development was interrupted in mid embryogenesis, involving defects related to dorsal closure and appendage formation. These phenotypes possibly result from the low levels of Broad-complex (BR-C) produced under JH-depleted conditions. This contrasts with holometabolan species, in which JH does not promote BR-C expression, which remains low during embryo development. Possibly, the stimulatory role of JH on BR-C expression and the morphogenetic functions of BR-C in hemimetabolan embryos were lost in holometabolan species. If so, this might have been a key driver for the evolution of holometabolan metamorphosis.

Smads and insect hemimetabolan metamorphosis.

In contrast with Drosophila melanogaster, practically nothing is known about the involvement of the TGF-ß signaling pathway in the metamorphosis of hemimetabolan insects. To partially fill this gap, we have studied the role of Smad factors in the metamorphosis of the German cockroach, Blattella germanica. In D. melanogaster, Mad is the canonical R-Smad of the BMP branch of the TGF-ß signaling pathway, Smox is the canonical R-Smad of the TGF-ß/Activin branch and Medea participates in both branches. In insects, metamorphosis is regulated by the MEKRE93 pathway, which starts with juvenile hormone (JH), whose signal is transduced by Methoprene-tolerant (Met), which stimulates the expression of Krüppel homolog 1 (Kr-h1) that acts to repress E93, the metamorphosis trigger. In B. germanica, metamorphosis is determined at the beginning of the sixth (final) nymphal instar (N6), when JH production ceases, the expression of Kr-h1 declines, and the transcription of E93 begins to increase. The RNAi of Mad, Smox and Medea in N6 of B. germanica reveals that the BMP branch of the TGF-ß signaling pathway regulates adult ecdysis and wing extension, mainly through regulating the expression of bursicon, whereas the TGF-ß/Activin branch contributes to increasing E93 and decreasing Kr-h1 at the beginning of N6, crucial for triggering adult morphogenesis, as well as to regulating the imaginal molt timing.

CREB-binding protein contributes to the regulation of endocrine and developmental pathways in insect hemimetabolan pre-metamorphosis.

BACKGROUND: CREB-binding protein (CBP) is a promiscuous transcriptional co-regulator. In insects, CBP has been studied in the fly Drosophila melanogaster, where it is known as Nejire. Studies in D. melanogaster have revealed that Nejire is involved in the regulation of many pathways during embryo development, especially in anterior/posterior polarity, through Hedgehog and Wingless signaling, and in dorsal/ventral patterning, through TGF-ß signaling. Regarding post-embryonic development, Nejire influences histone acetyl transferase activity on the ecdysone signaling pathway. METHODS AND RESULTS: Functional genomics studies using RNAi have shown that CBP contributes to the regulation of feeding and ecdysis during the pre-metamorphic nymphal instar of the cockroach Blattella germanica and is involved in TGF-ß, ecdysone, and MEKRE93 pathways, contributing to the activation of Kr-h1 and E93 expression. In D. melanogaster, Nejire's involvement in the ecdysone pathway in pre-metamorphic stages is conserved, whereas the TGF-ß pathway has only been described in the embryo. CBP role in ecdysis pathway and in the activation of Kr-h1 and E93 expression is described here for the first time. CONCLUSIONS: Studies in D. melanogaster may have been suggestive that CBP functions in insects are concentrated in the embryo. Results obtained in B. germanica indicate, however, that CBP have diverse and important functions in post-embryonic development and metamorphosis, especially regarding endocrine signaling. GENERAL SIGNIFICANCE: Further research into a higher diversity of models will probably reveal that the multiple post-embryonic roles of CBP observed in B. germanica are general in insects.