A DLG1-ARHGAP31-CDC42 axis is essential for the intestinal stem cell response to fluctuating niche Wnt signaling.

A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.

Evolutionary conservation of systemic and reversible amyloid aggregation.

In response to environmental stress, human cells have been shown to form reversible amyloid aggregates within the nucleus, termed amyloid bodies (A-bodies). These protective physiological structures share many of the biophysical characteristics associated with the pathological amyloids found in Alzheimer's and Parkinson's disease. Here, we show that A-bodies are evolutionarily conserved across the eukaryotic domain, with their detection in Drosophila melanogaster and Saccharomyces cerevisiae marking the first examples of these functional amyloids being induced outside of a cultured cell setting. The conditions triggering amyloidogenesis varied significantly among the species tested, with results indicating that A-body formation is a severe, but sublethal, stress response pathway that is tailored to the environmental norms of an organism. RNA-sequencing analyses demonstrate that the regulatory low-complexity long non-coding RNAs that drive A-body aggregation are both conserved and essential in human, mouse and chicken cells. Thus, the identification of these natural and reversible functional amyloids in a variety of evolutionarily diverse species highlights the physiological significance of this protein conformation, and will be informative in advancing our understanding of both functional and pathological amyloid aggregation events. This article has an associated First Person interview with the first author of the paper.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation. The process of basal stalk formation is not well understood, and we provide evidence that the cell intercalation is driven by actomyosin contractility of DE-Cadherin-adhered cells, leading to a column of disk-shaped cells exhibiting a novel radial cell polarity. Cell intercalation fails in Pak mutant ovaries, leading to abnormally wide basal stalks and consequently wide germaria with side-by-side cysts. We present evidence that Pak mutant germaria have extra FSCs, and we propose that contact of a germline cyst with the basal stalk in the pupal ovary contributes to FSC niche formation. The wide basal stalk in Pak mutants enables the formation of extra FSC niches which are mispositioned and yet functional, indicating that the FSC niche can be established in diverse locations.

Spatial orientation of different frequencies within the echolocation beam of a Tursiops truncatus and Pseudorca crassidens.

A two-dimensional array of 16 hydrophones was created to map the spatial distribution of different frequencies within the echolocation beam of a Tursiops truncatus and a Pseudorca crassidens. It was previously shown that both the Tursiops and Pseudorca only paid attention to frequencies between 29 and 42 kHz while echolocating. Both individuals tightly focused the 30 kHz frequency and the spatial location of the focus was consistently pointed toward the target. At 50 kHz the beam was less focused and less precisely pointed at the target. At 100 kHz the focus was often completely lost and was not pointed at the target. This indicates that these individuals actively focused the beam toward the target only in the frequency range they paid attention to. Frequencies outside this range were left unfocused and undirected. This focusing was probably achieved through sensorimotor control of the melon morphology and nasal air sacs. This indicates that both morphologically different species can control the spatial distribution of different frequency ranges within the echolocation beam to create consistent ensonation of desired targets.

High-frequency auditory filter shape for the Atlantic bottlenose dolphin.

High-frequency auditory filter shapes of an Atlantic bottlenose dolphin (Tursiops truncatus) were measured using a notched noise masking source centered on pure tone signals at frequencies of 40, 60, 80 and 100 kHz. A dolphin was trained to swim into a hoop station facing the noise/signal transducer located at a distance of 2 m. The dolphin's masked threshold was determined using an up-down staircase method as the width of the notched noise was randomly varied from 0, 0.2, 04, 0.6, and 0.8 times the test tone frequency. The masked threshold decreased as the width of the notched increased and less noise fell within the auditory filter associated with the test tone. The auditory filter shapes were approximated by fitting a roex (p,r(r)) function to the masked threshold results. A constant-Q value of 8.4 modeled the results within the frequency range of 40 to 100 kHz relatively well. However, between 60 and 100 kHz, the 3 dB bandwidth was relatively similar between 9.5 and 10 kHz, indicating a constant-bandwidth system in this frequency range The mean equivalent rectangular bandwidth calculated from the filter shape was approximately 16.0%, 17.0%, 13.6% and 11.3% of the tone frequencies of 40, 60, 80, and 100 kHz.

Single-lobed frequency-dependent beam shape in an echolocating false killer whale (Pseudorca crassidens).

Recent studies indicate some odontocetes may produce echolocation beams with a dual-lobed vertical structure. The shape of the odontocete echolocation beam was further investigated in a false killer whale performing an echolocation discrimination task. Clicks were recorded with an array of 16 hydrophones and frequency-dependent amplitude plots were constructed to assess beam shape. The majority of the echolocation clicks were single-lobed in structure with most energy located between 20 and 80 kHz. These data indicate the false killer whale does not produce a dual-lobed structure, as has been shown in bottlenose dolphins, which may be a function of lowered frequencies in the emitted signal due to hearing loss.

Genetic evidence for antagonism between Pak protein kinase and Rho1 small GTPase signaling in regulation of the actin cytoskeleton during Drosophila oogenesis.

During Drosophila oogenesis, basally localized F-actin bundles in the follicle cells covering the egg chamber drive its elongation along the anterior-posterior axis. The basal F-actin of the follicle cell is an attractive system for the genetic analysis of the regulation of the actin cytoskeleton, and results obtained in this system are likely to be broadly applicable in understanding tissue remodeling. Mutations in a number of genes, including that encoding the p21-activated kinase Pak, have been shown to disrupt organization of the basal F-actin and in turn affect egg chamber elongation. pak mutant egg chambers have disorganized F-actin distribution and remain spherical due to a failure to elongate. In a genetic screen to identify modifiers of the pak rounded egg chamber phenotype several second chromosome deficiencies were identified as suppressors. One suppressing deficiency removes the rho1 locus, and we determined using several rho1 alleles that removal of a single copy of rho1 can suppress the pak phenotype. Reduction of any component of the Rho1-activated actomyosin contractility pathway suppresses pak oogenesis defects, suggesting that Pak counteracts Rho1 signaling. There is ectopic myosin light chain phosphorylation in pak mutant follicle cell clones in elongating egg chambers, probably due at least in part to mislocalization of RhoGEF2, an activator of the Rho1 pathway. In early egg chambers, pak mutant follicle cells have reduced levels of myosin phosphorylation and we conclude that Pak both promotes and restricts myosin light chain phosphorylation in a temporally distinct manner during oogenesis.

The leading edge during dorsal closure as a model for epithelial plasticity: Pak is required for recruitment of the Scribble complex and septate junction formation.

Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.

Sonar-induced temporary hearing loss in dolphins.

There is increasing concern that human-produced ocean noise is adversely affecting marine mammals, as several recent cetacean mass strandings may have been caused by animals' interactions with naval 'mid-frequency' sonar. However, it has yet to be empirically demonstrated how sonar could induce these strandings or cause physiological effects. In controlled experimental studies, we show that mid-frequency sonar can induce temporary hearing loss in a bottlenose dolphin (Tursiops truncatus). Mild-behavioural alterations were also associated with the exposures. The auditory effects were induced only by repeated exposures to intense sonar pings with total sound exposure levels of 214 dB re: 1 microPa(2) s. Data support an increasing energy model to predict temporary noise-induced hearing loss and indicate that odontocete noise exposure effects bear trends similar to terrestrial mammals. Thus, sonar can induce physiological and behavioural effects in at least one species of odontocete; however, exposures must be of prolonged, high sound exposures levels to generate these effects.

Predicting temporary threshold shifts in a bottlenose dolphin (Tursiops truncatus): the effects of noise level and duration.

Noise levels in the ocean are increasing and are expected to affect marine mammals. To examine the auditory effects of noise on odontocetes, a bottlenose dolphin (Tursiops truncatus) was exposed to octave-band noise (4-8 kHz) of varying durations (<2-30 min) and sound pressures (130-178 dB re 1 microPa). Temporary threshold shift (TTS) occurrence was quantified in an effort to (i) determine the sound exposure levels (SELs) (dB re 1 microPa(2) s) that induce TTS and (ii) develop a model to predict TTS onset. Hearing thresholds were measured using auditory evoked potentials. If SEL was kept constant, significant shifts were induced by longer duration exposures but not for shorter exposures. Higher SELs were required to induce shifts in shorter duration exposures. The results did not support an equal-energy model to predict TTS onset. Rather, a logarithmic algorithm, which increased in sound energy as exposure duration decreased, was a better predictor of TTS. Recovery to baseline hearing thresholds was also logarithmic (approximately -1.8 dB/doubling of time) but indicated variability including faster recovery rates after greater shifts and longer recoveries necessary after longer duration exposures. The data reflected the complexity of TTS in mammals that should be taken into account when predicting odontocete TTS.

The perception of complex tones by a false killer whale (Pseudorca crassidens).

Complex tonal whistles are frequently produced by some odontocete species. However, no experimental evidence exists regarding the detection of complex tones or the discrimination of harmonic frequencies by a marine mammal. The objectives of this investigation were to examine the ability of a false killer whale to discriminate pure tones from complex tones and to determine the minimum intensity level of a harmonic tone required for the whale to make the discrimination. The study was conducted with a go/no-go modified staircase procedure. The different stimuli were complex tones with a fundamental frequency of 5 kHz with one to five harmonic frequencies. The results from this complex tone discrimination task demonstrated: (1) that the false killer whale was able to discriminate a 5 kHz pure tone from a complex tone with up to five harmonics, and (2) that discrimination thresholds or minimum intensity levels exist for each harmonic combination measured. These results indicate that both frequency level and harmonic content may have contributed to the false killer whale's discrimination of complex tones.

Atlantic bottlenose dolphin (Tursiops truncatus) hearing threshold for brief broadband signals.

The hearing sensitivity of an Atlantic bottlenose dolphin (Tursiops truncatus) to both pure tones and broadband signals simulating echoes from a 7.62-cm water-filled sphere was measured. Pure tones with frequencies between 40 and 140 kHz in increments of 20 kHz were measured along with broadband thresholds using a stimulus with a center frequency of 97.3 kHz and 88.2 kHz. The pure-tone thresholds were compared with the broadband thresholds by converting the pure-tone threshold intensity to energy flux density. The results indicated that dolphins can detect broadband signals slightly better than a pure-tone signal. The broadband results suggest that an echolocating bottlenose dolphin should be able to detect a 7.62-cm diameter water-filled sphere out to a range of 178 m in a quiet environment.