Pulsative venous return from the branchial vessels to the heart of the bivalve Mytilus galloprovincialis supports the constant-volume mechanism.

In bivalves and gastropods, ventricle contraction causes a negative pressure in the auricles and increases venous return from the afferent oblique vein (AOV): the constant-volume (CV) mechanism. The flow in the AOV should be a pulsative flow synchronized with the ventricular contraction. The flow in the heart and adjacent vessels of Mytilus galloprovincialis were measured by magnetic resonance imaging to confirm this hypothesis. Under a regular heartbeat, pulsative flows in the AOV and branchial vessels (BVs) were almost completely synchronized with the flow in the aorta, while filling of the ventricle was in the opposite phase. Flows in the BVs were directed to the posterior direction, and a pair of BVs in the gill axes (the efferent BVs) were connected to the AOV. Based on the images of the whole pathway of the AOV in an oblique slice, we confirmed that haemolymph flow was evoked from the efferent BVs and flow into the ventricle via the auricle was completed in a single heartbeat. Therefore, the walls of the AOV and BVs could resist negative transmural pressure caused by the ventricular contraction. In conclusion, the auricle, the AOV and the BVs, including the gill filaments, act as a suction pump. The pulsative venous return is driven by the negative pressure of the AOV as in the CV mechanism, and the negative pressure in the efferent BVs could draw haemolymph from the sinus via the gill and the afferent BVs. Therefore, Mytilus can start and stop its heartbeat as necessary.

Cohort-based multiscale analysis of hemodynamic-driven growth and remodeling of the embryonic pharyngeal arch arteries.

Growth and remodeling of the primitive pharyngeal arch artery (PAA) network into the extracardiac great vessels is poorly understood but a major source of clinically serious malformations. Undisrupted blood flow is required for normal PAA development, yet specific relationships between hemodynamics and remodeling remain largely unknown. Meeting this challenge is hindered by the common reductionist analysis of morphology to single idealized models, where in fact structural morphology varies substantially. Quantitative technical tools that allow tracking of morphological and hemodynamic changes in a population-based setting are essential to advancing our understanding of morphogenesis. Here, we have developed a methodological pipeline from high-resolution nano-computed tomography imaging and live-imaging flow measurements to multiscale pulsatile computational models. We combine experimental-based computational models of multiple PAAs to quantify hemodynamic forces in the rapidly morphing Hamburger Hamilton (HH) stage HH18, HH24 and HH26 embryos. We identify local morphological variation along the PAAs and their association with specific hemodynamic changes. Population-level mechano-morphogenic variability analysis is a powerful strategy for identifying stage-specific regions of well and poorly tolerated morphological and/or hemodynamic variation that may protect or initiate cardiovascular malformations.

An unexpected cyst of the ear lobule.

Branchial cleft anomalies (BCA) are among the most common congenital anomalies found in the pediatric head and neck. The embryology of these congenital anomalies is well understood, which allows clinicians to anticipate their diagnosis when a pediatric patient presents with a head or neck mass. The predictable anatomy of the various types of BCA allows for improved surgical planning to prevent recurrence and ensure complete resection. This report details an unusual location of a first BCA located in the ear lobule of a 10-month old male. There has been no documented first BCA at the ear lobule in the literature.

The Special Developmental Biology of Craniofacial Tissues Enables the Understanding of Oral and Maxillofacial Physiology and Diseases.

Maxillofacial hard tissues have several differences compared to bones of other localizations of the human body. These could be due to the different embryological development of the jaw bones compared to the extracranial skeleton. In particular, the immigration of neuroectodermally differentiated cells of the cranial neural crest (CNC) plays an important role. These cells differ from the mesenchymal structures of the extracranial skeleton. In the ontogenesis of the jaw bones, the development via the intermediate stage of the pharyngeal arches is another special developmental feature. The aim of this review was to illustrate how the development of maxillofacial hard tissues occurs via the cranial neural crest and pharyngeal arches, and what significance this could have for relevant pathologies in maxillofacial surgery, dentistry and orthodontic therapy. The pathogenesis of various growth anomalies and certain syndromes will also be discussed.

Progenitor targeting by adult stem cells in Ciona homeostasis, injury, and regeneration.

In the ascidian Ciona intestinalis, oral siphon amputation activates adult stem cell niches in the branchial sac to divide and dispatch migratory progenitor cells to a regeneration blastema at the site of injury. This study shows that progenitor cells derived from branchial sac stem cell niches have roles in homeostasis, wound repair, and regeneration of the siphons and neural complex (NC). During homeostasis, progenitor cells targeted the pharyngeal stigmata to replace ciliated cells involved in filter feeding. After individual or double siphon amputations, progenitor cells specifically targeted the oral or atrial siphons or both siphons, and were involved in the replacement of siphon circular muscle fibers. After oral siphon wounding, progenitor cells targeted the wound sites, and in some cases a supernumerary siphon was formed, although progenitor cell targeting did not predict the induction of supernumerary siphons. Following NC ablation, progenitor cells specifically targeted the regenerating NC, and supplied the precursors of new brain and neural gland cells. The tissues and organs targeted by branchial sac stem cells exhibited apoptosis during homeostasis and injury. It is concluded that branchial sac progenitor cells are multipotent and show targeting specificity that is correlated with apoptosis during homeostatic growth, tissue repair, and regeneration.

Skeletal kinematics of the hyoid arch in the suction-feeding shark Chiloscyllium plagiosum.

White-spotted bamboo sharks, Chiloscyllium plagiosum, generate strong suction-feeding pressures that rival the highest levels measured in ray-finned fishes. However, the hyostylic jaw suspension of these sharks is fundamentally different from the actinopterygian mechanism, including more mobile hyomandibulae, with the jaws and ceratohyal suspended from the hyomandibulae. Prior studies have proposed skeletal kinematics during feeding in orectolobid sharks from indirect measurements. Here, we tested these hypotheses using XROMM to measure cartilage motions directly. In agreement with prior hypotheses, we found extremely large retraction and depression of the ceratohyal, facilitated by large protraction and depression of the hyomandibula. Somewhat unexpectedly, XROMM also showed tremendous long-axis rotation (LAR) of both the ceratohyal and hyomandibula. This LAR likely increases the range of motion for the hyoid arch by keeping the elements properly articulated through their large arcs of motion. XROMM also confirmed that upper jaw protraction occurs before peak gape, similarly to actinopterygian suction feeders, but different from most other sharks in which jaw protrusion serves primarily to close the mouth. Early jaw protraction results from decoupling the rotations of the hyomandibula, with much of protraction occurring before peak gape with the other rotations lagging behind. In addition, the magnitudes of retraction and protraction of the hyoid elements are independent of the magnitude of depression, varying the shape of the mouth among feeding strikes. Hence, the large variation in suction-feeding behavior and performance may contribute to the wide dietary breadth of bamboo sharks.

Hcfc1b, a zebrafish ortholog of HCFC1, regulates craniofacial development by modulating mmachc expression.

Mutations in HCFC1 (MIM300019), have been recently associated with cblX (MIM309541), an X-linked, recessive disorder characterized by multiple congenital anomalies including craniofacial abnormalities. HCFC1 is a transcriptional co-regulator that modulates the expression of numerous downstream target genes including MMACHC, but it is not clear how these HCFC1 targets play a role in the clinical manifestations of cblX. To begin to elucidate the mechanism by which HCFC1 modulates disease phenotypes, we have carried out loss of function analyses in the developing zebrafish. Of the two HCFC1 orthologs in zebrafish, hcfc1a and hcfc1b, the loss of hcfc1b specifically results in defects in craniofacial development. Subsequent analysis revealed that hcfc1b regulates cranial neural crest cell differentiation and proliferation within the posterior pharyngeal arches. Further, the hcfc1b-mediated craniofacial abnormalities were rescued by expression of human MMACHC, a downstream target of HCFC1 that is aberrantly expressed in cblX. Furthermore, we tested distinct human HCFC1 mutations for their role in craniofacial development and demonstrated variable effects on MMACHC expression in humans and craniofacial development in zebrafish. Notably, several individuals with mutations in either HCFC1 or MMACHC have been reported to have mild to moderate facial dysmorphia. Thus, our data demonstrates that HCFC1 plays a role in craniofacial development, which is in part mediated through the regulation of MMACHC expression.

Musculin and TCF21 coordinate the maintenance of myogenic regulatory factor expression levels during mouse craniofacial development.

The specification of the skeletal muscle lineage during craniofacial development is dependent on the activity of MYF5 and MYOD, two members of the myogenic regulatory factor family. In the absence of MYF5 or MYOD there is not an overt muscle phenotype, whereas in the double Myf5;MyoD knockout branchiomeric myogenic precursors fail to be specified and skeletal muscle is not formed. The transcriptional regulation of Myf5 is controlled by a multitude of regulatory elements acting at different times and anatomical locations, with at least five operating in the branchial arches. By contrast, only two enhancers have been implicated in the regulation of MyoD. In this work, we characterize an enhancer element that drives Myf5 expression in the branchial arches from 9.5 days post-coitum and show that its activity in the context of the entire locus is dependent on two highly conserved E-boxes. These binding sites are required in a subset of Myf5-expressing cells including both progenitors and those which have entered the myogenic pathway. The correct levels of expression of Myf5 and MyoD result from activation by musculin and TCF21 through direct binding to specific enhancers. Consistent with this, we show that in the absence of musculin the timing of activation of Myf5 and MyoD is not affected but the expression levels are significantly reduced. Importantly, normal levels of Myf5 expression are restored at later stages, which might explain the absence of particular muscles in the Msc;Tcf21 double-knockout mice.

Rh protein expression in branchial neuroepithelial cells, and the role of ammonia in ventilatory control in fish.

Bill Milsom has made seminal contributions to our understanding of ventilatory control in a wide range of vertebrates. Teleosts are particularly interesting, because they produce a 3rd, potentially toxic respiratory gas (ammonia) in large amounts. Fish are well known to hyperventilate under high environmental ammonia (HEA), but only recently has the potential role of ammonia in normal ventilatory control been investigated. It is now clear that ammonia can act directly as a ventilatory stimulant in trout, independent of its effects on acid-base balance. Even in ureotelic dogfish sharks, acute elevations in ammonia cause increases in ventilation. Peripherally, the detection of elevated ammonia resides in gill arches I and II in trout, and in vitro, neuroepithelial cells (NECs) from these arches are sensitive to ammonia, responding with elevations in intracellular Ca(2+) ([Ca(2+)]i). Centrally, hyperventilatory responses to ammonia correlate more closely with concentrations of ammonia in the brain than in plasma or CSF. After chronic HEA exposure, ventilatory responsiveness to ammonia is lost, associated with both an attenuation of the [Ca(2+)]i response in NECs, and the absence of elevation in brain ammonia concentration. Chronic exposure to HEA also causes increases in the mRNA expression of several Rh proteins (ammonia-conductive channels) in both brain and gills. "Single cell" PCR techniques have been used to isolate the individual responses of NECs versus other gill cell types. We suggest several circumstances (post-feeding, post-exercise) where the role of ammonia as a ventilatory stimulant may have adaptive benefits for O2 uptake in fish.

Protocadherin PAPC is expressed in the CNC and can compensate for the loss of PCNS.

Protocadherins represent the biggest subgroup within the cadherin superfamily of transmembrane glycoproteins. In contrast to classical type I cadherins, protocadherins in general exhibit only moderate adhesive activity. During embryogenesis, they are involved in cell signaling and regulate diverse morphogenetic processes, including morphogenetic movements during gastrulation and neural crest migration. The two protocadherins paraxial protocadherin (PAPC) and axial protocadherin (AXPC) are indispensable for proper gastrulation movements in Xenopus and zebrafish. The closest relative PCNS instead, is required for neural crest and somite formation. Here, we show that cranial neural crest (CNC) cells in addition to PCNS express PAPC, but not AXPC. Overexpression of PAPC resulted in comparable migration defects as knockdown of PCNS. Moreover, reconstitution experiments revealed that PAPC is able to replace PCNS in CNC cells, indicating that both protocadherins can regulate CNC migration.

Anatomical and physiological studies of bigheaded carps demonstrate that the epibranchial organ functions as a pharyngeal taste organ.

The epibranchial organ (EO) is an enigmatic tubular organ found in the pharyngeal cavity of many filter-feeding fishes. We investigated whether it might function as a taste organ that mediates aggregation and ingestion of planktonic food within the buccal cavity. The EO and associated structures of bighead and silver carps, two successful and invasive planktivorous fishes, were examined using histological and electrophysiological techniques. Both species possess finely structured gill rakers that extend directly via a series of protrusions into each of the four blind canals which are organized as the muscular EO, suggesting that the gill rakers and EO probably function in an integrated manner. Both the interior and exterior surfaces of the EOs of both species are covered with high densities of taste buds and solitary chemosensory cells (SCCs) as well as mucous cells. Conversely, taste buds are scarce in both the buccal cavities and external portions of the head and mouth of both species. Electrophysiological recordings from a caudal branch of the vagus nerve (cranial nerve X) found to innervate the EO showed it to be sensitive to chemicals found in a planktonic diet. l-Amino acids accounted for some, but not all of the neural activity. We conclude that taste buds and SCCs located on the EO and gill rakers probably serve to chemically detect food particles, which the EO then aggregates by mucus secretion before eventually expelling them onto the floor of the pharynx for ingestion. This specialized, pharyngeal chemosensory structure may explain the feeding success of these, and perhaps other planktivorous, filter-feeding fishes.

New insights into the role of the pharyngeal jaw apparatus in the sound-producing mechanism of Haemulon flavolineatum (Haemulidae).

Grunts are fish that are well known to vocalize, but how they produce their grunting sounds has not been clearly identified. In addition to characterizing acoustic signals and hearing in the French grunt Haemulon flavolineatum, the present study investigates the sound-production mechanism of this species by means of high-speed X-ray videos and scanning electron microscopy of the pharyngeal jaw apparatus. Vocalizations consist of a series of stridulatory sounds: grunts lasting ~47 ms with a mean period of 155 ms and a dominant frequency of ~700 Hz. Auditory capacity was determined to range from 100 to 600 Hz, with greatest sensitivity at 300 Hz (105.0±11.8 dB re. 1 µPa). This suggests that hearing is not tuned exclusively to detect the sounds of conspecifics. High-speed X-ray videos revealed how pharyngeal jaws move during sound production. Traces of erosion on teeth in the fourth ceratobranchial arch suggest that they are also involved in sound production. The similarity of motor patterns of the upper and lower pharyngeal jaws between food processing and sound production indicates that calling is an exaptation of the food-processing mechanism.

Effects of salinity on metabolic rate and branchial expression of genes involved in ion transport and metabolism in Mozambique tilapia (Oreochromis mossambicus).

This study investigated the effects of two rearing salinities, and acute salinity transfer, on the energetic costs of osmoregulation and the expression of metabolic and osmoregulatory genes in the gill of Mozambique tilapia. Using automated, intermittent-flow respirometry, measured standard metabolic rates (SMRs) of tilapia reared in seawater (SW, 130 mg O2 kg⁻¹ h⁻¹) were greater than those reared in fresh water (FW, 103 mg O2 kg⁻¹ h⁻¹), when normalized to a common mass of 0.05 kg and at 25±1°C. Transfer from FW to 75% SW increased SMR within 18h, to levels similar to SW-reared fish, while transfer from SW to FW decreased SMR to levels similar to FW-reared fish. Branchial gene expression of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC), an indicator of SW-type mitochondria-rich (MR) cells, was positively correlated with SMR, while Na⁺-Cl⁻ cotransporter (NCC), an indicator of FW-type MR cells, was negatively correlated. Principal Components Analysis also revealed that branchial expression of cytochrome c oxidase subunit IV (COX-IV), glycogen phosphorylase (GP), and a putative mitochondrial biogenesis regulator in fish, peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), were correlated with a higher SMR, plasma osmolality, and environmental salinity, while expression of glycogen synthase (GS), PGC-1ß, and nuclear respiratory factor 1 (NRF-1) had negative correlations. These results suggest that the energetic costs of osmoregulation are higher in SW than in FW, which may be related to the salinity-dependent differences in osmoregulatory mechanisms found in the gills of Mozambique tilapia.

Osmoregulation and branchial plasticity after acute freshwater transfer in red drum, Sciaenops ocellatus.

Red drum, Sciaenops ocellatus, is an estuarine-dependent fish species commonly found in the Gulf of Mexico and along the coast of the southeastern United States. This economically important species has demonstrated freshwater tolerance; however, the physiological mechanisms and costs related to freshwater exposure remain poorly understood. The current study therefore investigated the physiological response of red drum using an acute freshwater transfer protocol. Plasma osmolality, Cl⁻, Mg²âº and Ca²âº were all significantly reduced by 24h post-transfer; Cl⁻ and Mg²âº recovered to control levels by 7days post-transfer. No effect of transfer was observed on muscle water content; however, muscle Cl⁻ was significantly reduced. Interestingly, plasma and muscle Na⁺ content was unaffected by freshwater transfer. Intestinal fluid was absent by 24h post-transfer indicating cessation of drinking. Branchial gene expression analysis showed that both CFTR and NKCC1 exhibited significant down-regulation at 8 and 24h post-transfer, respectively, although transfer had no impact on NHE2, NHE3 or Na⁺, K⁺ ATPase (NKA) activity. These general findings are supported by immunohistochemical analysis, which revealed no apparent NKCC containing cells in the gills at 7days post transfer while NKA cells localization was unaffected. The results of the current study suggest that red drum can effectively regulate Na⁺ balance upon freshwater exposure using already present Na⁺ uptake pathways while also down-regulating ion excretion mechanisms.

An essential requirement for ß1 integrin in the assembly of extracellular matrix proteins within the vascular wall.

ß1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of ß1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that ß1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of ß1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for ß1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that ß1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of ß1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.

hand2 and Dlx genes specify dorsal, intermediate and ventral domains within zebrafish pharyngeal arches.

The ventrally expressed secreted polypeptide endothelin1 (Edn1) patterns the skeleton derived from the first two pharyngeal arches into dorsal, intermediate and ventral domains. Edn1 activates expression of many genes, including hand2 and Dlx genes. We wanted to know how hand2/Dlx genes might generate distinct domain identities. Here, we show that differential expression of hand2 and Dlx genes delineates domain boundaries before and during cartilage morphogenesis. Knockdown of the broadly expressed genes dlx1a and dlx2a results in both dorsal and intermediate defects, whereas knockdown of three intermediate-domain restricted genes dlx3b, dlx4b and dlx5a results in intermediate-domain-specific defects. The ventrally expressed gene hand2 patterns ventral identity, in part by repressing dlx3b/4b/5a. The jaw joint is an intermediate-domain structure that expresses nkx3.2 and a more general joint marker, trps1. The jaw joint expression of trps1 and nkx3.2 requires dlx3b/4b/5a function, and expands in hand2 mutants. Both hand2 and dlx3b/4b/5a repress dorsal patterning markers. Collectively, our work indicates that the expression and function of hand2 and Dlx genes specify major patterning domains along the dorsoventral axis of zebrafish pharyngeal arches.

Comparative analysis of Neph gene expression in mouse and chicken development.

Neph proteins are evolutionarily conserved members of the immunoglobulin superfamily of adhesion proteins and regulate morphogenesis and patterning of different tissues. They share a common protein structure consisting of extracellular immunoglobulin-like domains, a transmembrane region, and a carboxyl terminal cytoplasmic tail required for signaling. Neph orthologs have been widely characterized in invertebrates where they mediate such diverse processes as neural development, synaptogenesis, or myoblast fusion. Vertebrate Neph proteins have been described first at the glomerular filtration barrier of the kidney. Recently, there has been accumulating evidence suggesting a function of Neph proteins also outside the kidney. Here we demonstrate that Neph1, Neph2, and Neph3 are expressed differentially in various tissues during ontogenesis in mouse and chicken. Neph1 and Neph2 were found to be amply expressed in the central nervous system while Neph3 expression remained localized to the cerebellum anlage and the spinal cord. Outside the nervous system, Neph mRNAs were also differentially expressed in branchial arches, somites, heart, lung bud, and apical ectodermal ridge. Our findings support the concept that vertebrate Neph proteins, similarly to their Drosophila and C. elegans orthologs, provide guidance cues for cell recognition and tissue patterning in various organs which may open interesting perspectives for future research on Neph1-3 controlled morphogenesis.

Plasticity of neural crest-placode interaction in the developing visceral nervous system.

The reciprocal relationship between rhombomere (r)-derived cranial neural crest (NC) and epibranchial placodal cells derived from the adjacent branchial arch is critical for visceral motor and sensory gangliogenesis, respectively. However, it is unknown whether the positional match between these neurogenic precursors is hard-wired along the anterior-posterior (A/P) axis. Here, we use the interaction between r4-derived NC and epibranchial placode-derived geniculate ganglion as a model to address this issue. In Hoxa1(-/-) b1(-/-) embryos, r2 NC compensates for the loss of r4 NC. Specifically, a population of r2 NC cells is redirected toward the geniculate ganglion, where they differentiate into postganglionic (motor) neurons. Reciprocally, the inward migration of the geniculate ganglion is associated with r2 NC. The ability of NC and placodal cells to, respectively, differentiate and migrate despite a positional mismatch along the A/P axis reflects the plasticity in the relationship between the two neurogenic precursors of the vertebrate head.

Bottles as models: predicting the effects of varying swimming speed and morphology on size selectivity and filtering efficiency in fishes.

We created physical models based on the morphology of ram suspension-feeding fishes to better understand the roles morphology and swimming speed play in particle retention, size selectivity and filtration efficiency during feeding events. We varied the buccal length, flow speed and architecture of the gills slits, including the number, size, orientation and pore size/permeability, in our models. Models were placed in a recirculating flow tank with slightly negatively buoyant plankton-like particles (~20-2000 µm) collected at the simulated esophagus and gill rakers to locate the highest density of particle accumulation. Particles were captured through sieve filtration, direct interception and inertial impaction. Changing the number of gill slits resulted in a change in the filtration mechanism of particles from a bimodal filter, with very small (≤ 50 µm) and very large (>1000 µm) particles collected, to a filter that captured medium-sized particles (101-1000 µm). The number of particles collected on the gill rakers increased with flow speed and skewed the size distribution towards smaller particles (51-500 µm). Small pore sizes (105 and 200 µm mesh size) had the highest filtration efficiencies, presumably because sieve filtration played a significant role. We used our model to make predictions about the filtering capacity and efficiency of neonatal whale sharks. These results suggest that the filtration mechanics of suspension feeding are closely linked to an animal's swimming speed and the structural design of the buccal cavity and gill slits.

Placodal sensory ganglia coordinate the formation of the cranial visceral motor pathway.

The parasympathetic reflex circuit is controlled by three basic neurons. In the vertebrate head, the sensory, and pre- and postganglionic neurons that comprise each circuit have stereotypic positions along the anteroposterior (AP) axis, suggesting that the circuit arises from a common developmental plan. Here, we show that precursors of the VIIth circuit are initially aligned along the AP axis, where the placode-derived sensory neurons provide a critical "guidepost" through which preganglionic axons and their neural crest-derived postganglionic targets navigate before reaching their distant target sites. In the absence of the placodal sensory ganglion, preganglionic axons terminate and the neural crest fated for postganglionic neurons undergo apoptosis at the site normally occupied by the placodal sensory ganglion. The stereotypic organization of the parasympathetic cranial sensory-motor circuit thus emerges from the initial alignment of its precursors along the AP axis, with the placodal sensory ganglion coordinating the formation of the motor pathway.