Photoreceptor calyceal processes accompany the developing outer segment, adopting a stable length despite a dynamic core.

Vertebrate photoreceptors detect light through a large cilium-based outer segment, which is filled with photopigment-laden membranous discs. Surrounding the base of the outer segment are microvilli-like calyceal processes (CPs). Although CP disruption has been associated with altered outer segment morphology and photoreceptor degeneration, the role of the CPs remains elusive. Here, we used zebrafish as a model to characterize CPs. We quantified CP parameters and report a strong disparity in outer segment coverage between photoreceptor subtypes. CP length is stable across light and dark conditions, yet heat-shock inducible expression of tagged actin revealed rapid turnover of the CP actin core. Detailed imaging of the embryonic retina uncovered substantial remodeling of the developing photoreceptor apical surface, including a transition from dynamic tangential processes to vertically oriented CPs immediately prior to outer segment formation. Remarkably, we also found a direct connection between apical extensions of the Müller glia and retinal pigment epithelium, arranged as bundles around the ultraviolet sensitive cones. In summary, our data characterize the structure, development and surrounding environment of photoreceptor microvilli in the zebrafish retina.

Fatty infiltration of gastrocnemius-soleus muscle complex: Considerations for myosteatosis rehabilitation.

Although previous studies have reported fatty infiltration of the gastrocnemius-soleus complex, little is known about the volumetric distribution and patterns of fatty infiltration. The purpose of this anatomical study was to document and quantify the frequency, distribution, and pattern of fatty infiltration of the gastrocnemius-soleus complex. One hundred formalin-embalmed specimens (mean age 78.1 ± 12.3 years; 48F/52M) were serially dissected to document the frequency, distribution, and pattern of fatty infiltration in the medial and lateral heads of gastrocnemius and soleus muscles. Fatty infiltration was found in 23% of specimens, 13 unilaterally (8F/5M) and 10 (5M/5F) bilaterally. The fatty infiltration process was observed to begin medially from the medial aspect of the medial head of gastrocnemius and medial margin of soleus and then progressed laterally throughout the medial head of gastrocnemius and the marginal, anterior, and posterior soleus. The lateral head of gastrocnemius remained primarily muscular in all specimens. Microscopically, the pattern of infiltration was demonstrated as intramuscular with intact aponeuroses, and septa. The remaining endo-, peri-, and epimysium preserved the overall contour of the gastrocnemius-soleus complex, even in cases of significant fatty replacement. Since the external contour of the calf is preserved, the presence of fatty infiltration may be underdiagnosed in the clinic without imaging. Myosteatosis is associated with gait and balance challenges in the elderly, which can impact quality of life and result in increased risk of falling. The findings of the study have implications in the rehabilitation management of elderly patients with sarcopenia and myosteatosis.

SOMANZ position statement for the investigation and management of sepsis in pregnancy 2023.

BACKGROUND: The Society of Australia and New Zealand (SOMANZ) published its first sepsis in pregnancy and the postpartum period guideline in 2017 (Aust N Z J Obstet Gynaecol, 57, 2017, 540). In the intervening 6 years, maternal mortality from sepsis has remained static. AIMS: To update clinical practice with a review of the subsequent literature. In particular, to review the definition and screening tools for the diagnosis of sepsis. MATERIALS AND METHODS: A multi-disciplinary group of clinicians with experience in all aspects of the care of pregnant women analysed the clinical evidence according to the GRADE (Grading of Recommendations Assessment, Development and Evaluation) system following searches of Cochrane, Medline and EMBASE. Where there were conflicting views, the authors reviewed the topic and came to a consensus. All authors reviewed the final position statement. RESULTS: This position statement has abandoned the use of the quick Sequential Organ Failure Assessment score (qSOFA) score to diagnose sepsis due to its poor performance in clinical practice. Whilst New Zealand has a national maternity observation chart, in Australia maternity early warning system charts and vital sign cut-offs differ between states. Rapid recognition, early antimicrobials and involvement of senior staff remain essential factors to improving outcomes. CONCLUSION: Ongoing research is required to discover and validate tools to recognize and diagnose sepsis in pregnancy. Australia should follow New Zealand and have a single national maternity early warning system observation chart.

The morphological and functional diversity of apical microvilli.

Sensory neurons use specialized apical processes to perceive external stimuli and monitor internal body conditions. The apical apparatus can include cilia, microvilli, or both, and is adapted for the functions of the particular cell type. Photoreceptors detect light through a large, modified cilium (outer segment), that is supported by a surrounding ring of microvilli-like calyceal processes (CPs). Although first reported 150 years ago, CPs remain poorly understood. As a basis for future study, we therefore conducted a review of existing literature about sensory cell microvilli, which can act either as the primary sensory detector or as support for a cilia-based detector. While all microvilli are finger-like cellular protrusions with an actin core, the processes vary across cell types in size, number, arrangement, dynamics, and function. We summarize the current state of knowledge about CPs and the characteristics of the microvilli found on inner ear hair cells (stereocilia) and cerebral spinal fluid-contacting neurons, with comparisons to the brush border of the intestinal and renal epithelia. The structure, stability, and dynamics of the actin core are regulated by a complement of actin-binding proteins, which includes both common components and unique features when compared across cell types. Further, microvilli are often supported by lateral links, a glycocalyx, and a defined extracellular matrix, each adapted to the function and environment of the cell. Our comparison of microvillar features will inform further research into how CPs support photoreceptor function, and also provide a general basis for investigations into the structure and functions of apical microvilli found on sensory neurons.

Electroretinogram analysis of zebrafish retinal function across development.

PURPOSE: The electroretinogram (ERG) is a powerful approach for investigating visual function in zebrafish ocular disease models. However, complexity, cost, and a literature gap present as significant barriers for the introduction of this technology to new zebrafish laboratories. Here, we introduce a simplified and effective method to obtain zebrafish ERGs. METHODS: In-house assembled recording electrodes and a custom 3D-printed platform were used to gather high-quality and consistent ERG data from zebrafish at 3 developmental timepoints-larval, juvenile, and adult. Fish were tested under both scotopic (dark-adapted) and photopic (light-adapted) conditions to differentiate between the rod and cone systems, respectively. RESULTS: Robust ERG waveforms across all developmental timepoints were obtained using the methodology presented here. We observed an overall increase in signal amplitude as development progressed, reflecting maturation of the zebrafish retina. Oscillatory potentials could also be isolated from the generated waveforms. CONCLUSIONS: This simplified approach to the zebrafish ERG can generate waveforms comparable to the existing approaches and helps reduce barriers for zebrafish laboratories studying ocular development and disease.

Studying the Tumor Microenvironment in Zebrafish.

The tumor microenvironment significantly contributes to tumor initiation, progression, neo-angiogenesis, and metastasis, and a better understanding of the role of the different cellular players would facilitate the development of novel therapeutic strategies for cancer treatment. Towards this goal, intravital imaging is a powerful method to unravel interaction partners of tumor cells. Among vertebrate model organisms, zebrafish is uniquely suited for in vivo imaging studies. In recent years zebrafish has also become a valuable model in cancer research. In this chapter, we will summarize, how zebrafish has been used to characterize cells of the tumor microenvironment. We will cover both genetically engineered cancer models and xenograft models in zebrafish. The majority of work has been done on the role of innate immune cells and their role during tumor initiation and metastasis, but we will also cover studies focusing on adipocytes, fibroblasts, and endothelial cells. Taken together, we will highlight the versatile use of the zebrafish model for in vivo tumor microenvironment studies.

Morphogenetic defects underlie Superior Coloboma, a newly identified closure disorder of the dorsal eye.

The eye primordium arises as a lateral outgrowth of the forebrain, with a transient fissure on the inferior side of the optic cup providing an entry point for developing blood vessels. Incomplete closure of the inferior ocular fissure results in coloboma, a disease characterized by gaps in the inferior eye and recognized as a significant cause of pediatric blindness. Here, we identify eight patients with defects in tissues of the superior eye, a congenital disorder that we term superior coloboma. The embryonic origin of superior coloboma could not be explained by conventional models of eye development, leading us to reanalyze morphogenesis of the dorsal eye. Our studies revealed the presence of the superior ocular sulcus (SOS), a transient division of the dorsal eye conserved across fish, chick, and mouse. Exome sequencing of superior coloboma patients identified rare variants in a Bone Morphogenetic Protein (Bmp) receptor (BMPR1A) and T-box transcription factor (TBX2). Consistent with this, we find sulcus closure defects in zebrafish lacking Bmp signaling or Tbx2b. In addition, loss of dorsal ocular Bmp is rescued by concomitant suppression of the ventral-specific Hedgehog pathway, arguing that sulcus closure is dependent on dorsal-ventral eye patterning cues. The superior ocular sulcus acts as a conduit for blood vessels, with altered sulcus closure resulting in inappropriate connections between the hyaloid and superficial vascular systems. Together, our findings explain the existence of superior coloboma, a congenital ocular anomaly resulting from aberrant morphogenesis of a developmental structure.

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation.

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.

Zebrafish jam-b2 Gal4-enhancer trap line recapitulates endogenous jam-b2 expression in extraocular muscles.

BACKGROUND: Members of the junctional adhesion molecule (JAM) family function as cell adhesion molecules and cell surface receptors. The zebrafish genome contains six different jam genes, and jam-b and jam-c were shown to be essential for myoblast fusion during skeletal muscle development. However, little is known about jam-b2 expression and function. RESULTS: We isolated the cDNA of zebrafish jam-b2. jam-b2 is expressed specifically in extraocular muscles (EOMs), jaw muscles, and pectoral fins in zebrafish larvae, but not in trunk muscles. The identified jam-b2 expression pattern is supported by the analysis of a zebrafish Gal4-enhancer trap line, in which the coding sequence of the transcriptional activator KalTA4 together with a Gal4-dependent UAS-mCherry expression cassette was inserted into the jam-b2 locus. Intercrosses with an UAS:EGFP strain proves the possibility for targeting transgene expression to EOMs, jaw muscles and fins. Finally, we characterized the concerted contraction pattern of EOMs in larvae performing an optokinetic response. CONCLUSIONS: The expression pattern of jam-b2 suggests that it may contribute different properties to EOMs, jaw muscles, and pectoral fins. The jam-b2:KalTA4-UAS-mCherry transgenic strain serves a dual role as both a reporter for these muscles and as a valuable genetic tool for targeting transgene expression to EOMs.

The role of Zic transcription factors in regulating hindbrain retinoic acid signaling.

BACKGROUND: The reiterated architecture of cranial motor neurons aligns with the segmented structure of the embryonic vertebrate hindbrain. Anterior-posterior identity of cranial motor neurons depends, in part, on retinoic acid signaling levels. The early vertebrate embryo maintains a balance between retinoic acid synthetic and degradative zones on the basis of reciprocal expression domains of the retinoic acid synthesis gene aldhehyde dehydrogenase 1a2 (aldh1a2) posteriorly and the oxidative gene cytochrome p450 type 26a1 (cyp26a1) in the forebrain, midbrain, and anterior hindbrain. RESULTS: This manuscript investigates the role of zinc finger of the cerebellum (zic) transcription factors in regulating levels of retinoic acid and differentiation of cranial motor neurons. Depletion of zebrafish Zic2a and Zic2b results in a strong downregulation of aldh1a2 expression and a concomitant reduction in activity of a retinoid-dependent transgene. The vagal motor neuron phenotype caused by loss of Zic2a/2b mimics a depletion of Aldh1a2 and is rescued by exogenously supplied retinoic acid. CONCLUSION: Zic transcription factors function in patterning hindbrain motor neurons through their regulation of embryonic retinoic acid signaling.

A cell atlas of microbe-responsive processes in the zebrafish intestine.

Gut microbial products direct growth, differentiation, and development in animal hosts. However, we lack system-wide understanding of cell-specific responses to the microbiome. We profiled cell transcriptomes from the intestine, and associated tissue, of zebrafish larvae raised in the presence or absence of a microbiome. We uncovered extensive cellular heterogeneity in the conventional zebrafish intestinal epithelium, including previously undescribed cell types with known mammalian homologs. By comparing conventional to germ-free profiles, we mapped microbial impacts on transcriptional activity in each cell population. We revealed intricate degrees of cellular specificity in host responses to the microbiome that included regulatory effects on patterning and on metabolic and immune activity. For example, we showed that the absence of microbes hindered pro-angiogenic signals in the developing vasculature, causing impaired intestinal vascularization. Our work provides a high-resolution atlas of intestinal cellular composition in the developing fish gut and details the effects of the microbiome on each cell type.

Evaluation of online interprofessional simulation workshops for obstetric and neonatal emergencies.

Objectives: To explore student perceptions of learning and interprofessional aspects of obstetric and neonatal emergencies through online simulation-based workshops. Methods: This qualitative study was conducted at Monash University, Australia. Data were obtained from six separate online Obstetric Neonatal Emergency Simulation workshops held between May 2020 and August 2021. A total of 385 students attended and were invited to participate in the study by completing an online survey two-three weeks later. Of the attendees, 144 students completed the survey (95 medical, 45 midwifery), equating to a response rate of 37%. Survey responses were downloaded from online survey platform and separated into medical and midwifery responses. Thematic analysis of data was performed using a coding framework, resulting in development of themes and subthemes. Results: Main themes were adaptability, connectivism, preparedness for practice, experiential learning, learning through modelling and dynamics of online interaction. Students reported that online workshop was a useful alternative method to experience simulation-based learning, increase their readiness for clinical practice and foster positive interprofessional relationships. Consistent with existing literature evaluating similar in-person programs, midwifery students were most interested in interprofessional interaction (predominant theme: dynamics of online interaction), whilst medical students were more concerned with developing clinical skills (predominant themes: learning through modelling, experiential learning). Conclusions: Online learning may be a useful and convenient way of delivering interprofessional simulation-based education during the pandemic, in remote areas and as an adjunct to in-person teaching. Future studies should evaluate the impact of online learning with a mixed methods study and in comparison, to in-person programs.

Zebrafish and inherited photoreceptor disease: Models and insights.

Photoreceptor dysfunctions and degenerative diseases are significant causes of vision loss in patients, with few effective treatments available. Targeted interventions to prevent or reverse photoreceptor-related vision loss are not possible without a thorough understanding of the underlying mechanism leading to disease, which is exceedingly difficult to accomplish in the human system. Cone diseases are particularly challenging to model, as some popular genetically modifiable model animals are nocturnal with a rod-dominant visual system and cones that have dissimilarities to human cones. As a result, cone diseases, which affect visual acuity, colour perception, and central vision in patients, are generally poorly understood in terms of pathology and mechanism. Zebrafish (Danio rerio) provide the opportunity to model photoreceptor diseases in a diurnal vertebrate with a cone-rich retina which develops many macular degeneration-like pathologies. Zebrafish undergo external development, allowing early-onset retinal diseases to be detected and studied, and many ophthalmic tools are available for zebrafish visual assessment during development and adulthood. There are numerous zebrafish models of photoreceptor disease, spanning the various types of photoreceptor disease (developmental, rod, cone, and mixed photoreceptor diseases) and genetic/molecular cause. In this review, we explore the features of zebrafish that make them uniquely poised to model cone diseases, summarize the established zebrafish models of inherited photoreceptor disease, and discuss how disease in these models compares to the human presentation, where applicable. Further, we highlight the contributions of these zebrafish models to our understanding of photoreceptor biology and disease, and discuss future directions for utilising and investigating these diverse models.

The inner junction protein CFAP20 functions in motile and non-motile cilia and is critical for vision.

Motile and non-motile cilia are associated with mutually-exclusive genetic disorders. Motile cilia propel sperm or extracellular fluids, and their dysfunction causes primary ciliary dyskinesia. Non-motile cilia serve as sensory/signalling antennae on most cell types, and their disruption causes single-organ ciliopathies such as retinopathies or multi-system syndromes. CFAP20 is a ciliopathy candidate known to modulate motile cilia in unicellular eukaryotes. We demonstrate that in zebrafish, cfap20 is required for motile cilia function, and in C. elegans, CFAP-20 maintains the structural integrity of non-motile cilia inner junctions, influencing sensory-dependent signalling and development. Human patients and zebrafish with CFAP20 mutations both exhibit retinal dystrophy. Hence, CFAP20 functions within a structural/functional hub centered on the inner junction that is shared between motile and non-motile cilia, and is distinct from other ciliopathy-associated domains or macromolecular complexes. Our findings suggest an uncharacterised pathomechanism for retinal dystrophy, and potentially for motile and non-motile ciliopathies in general.

Quasi-Experimental study of effects of lighting on rest, activity and melatonin in postpartum women.

OBJECTIVES: to compare the parameters of the activity/rest cycle of early postpartum breastfeeding women under a controlled and uncontrolled long wavelength ray light regimen. METHODS: quasi-experimental study with breastfeeding women and their babies during postnatal rooming-in, São Paulo, Brazil. Participants were allocated to either an experimental (intervention) or a comparison group. The intervention involved exposure of the woman in a controlled room with artificial long wavelength ray light at night. Each woman's level of 6-sulfatoxymelatonin at 24 hours and activity/rest times was analyzed. RESULTS: the mean activity/rest times of women in the experimental and comparison groups were similar. The mean percentages of total load of 6-sulfatoxymelatonin during the day and night were similar (p=0.09). At 24 hours, the experimental group presented a significantly lower mean percentage of total load compared to the comparison group (p=0.04). CONCLUSIONS: women who stayed in the room with long-wavelength artificial light showed no difference in activity/rest and 6-sulfatoxymelatonin levels in the early postpartum period.

LIMK1 acts downstream of BMP signaling in developing retinal ganglion cell axons but not dendrites.

The actin cytoskeleton inside extending axonal and dendritic processes must undergo continuous assembly and disassembly. Some extrinsic factors modulate actin turnover through controlling the activity of LIM kinase 1 (LIMK1), which phosphorylates and inactivates the actin depolymerizing factor cofilin. Here, we for the first time examine the function and regulation of LIMK1 in vivo in the vertebrate nervous system. Upon expression of wildtype or kinase-dead forms of the protein, dendrite growth by Xenopus retinal ganglion cells (RGCs) was unchanged. In contrast, maintaining a low, but significant level, of LIMK1 function in the RGC axon is critical for proper extension. Interestingly, bone morphogenetic protein receptor II (BMPRII) is a major regulator of LIMK1 in extending RGC axons, as expression of a BMPRII lacking the LIMK1 binding region caused a dramatic shortening of the axons. Previously, we found that BMPRIIs stimulate dendrite initiation in vivo. Thus, the fact that manipulation of LIMK1 activity failed to alter dendrite growth suggests that BMPs may activate distinct signalling pathways in axons and dendrites.

Progressive Photoreceptor Dysfunction and Age-Related Macular Degeneration-Like Features in rp1l1 Mutant Zebrafish.

Photoreceptor disease results in irreparable vision loss and blindness, which has a dramatic impact on quality of life. Pathogenic mutations in RP1L1 lead to photoreceptor degenerations such as occult macular dystrophy and retinitis pigmentosa. RP1L1 is a component of the photoreceptor axoneme, the backbone structure of the photoreceptor's light-sensing outer segment. We generated an rp1l1 zebrafish mutant using CRISPR/Cas9 genome editing. Mutant animals had progressive photoreceptor functional defects as determined by electrophysiological assessment. Optical coherence tomography showed gaps in the photoreceptor layer, disrupted photoreceptor mosaics, and thinner retinas. Mutant retinas had disorganized photoreceptor outer segments and lipid-rich subretinal drusenoid deposits between the photoreceptors and retinal pigment epithelium. Our mutant is a novel model of RP1L1-associated photoreceptor disease and the first zebrafish model of photoreceptor degeneration with reported subretinal drusenoid deposits, a feature of age-related macular degeneration.

Abnormal Cone and Rod Photoreceptor Morphogenesis in gdf6a Mutant Zebrafish.

Purpose: Analysis of photoreceptor morphology and gene expression in mispatterned eyes of zebrafish growth differentiation factor 6a (gdf6a) mutants. Methods: Rod and cone photoreceptors were compared between gdf6a mutant and control zebrafish from larval to late adult stages using transgenic labels, immunofluorescence, and confocal microscopy, as well as by transmission electron microscopy. To compare transcriptomes between larval gdf6a mutant and control zebrafish, RNA-Seq was performed on isolated eyes. Results: Although rod and cone photoreceptors differentiate in gdf6a mutant zebrafish, the cells display aberrant growth and morphology. The cone outer segments, the light-detecting sensory endings, are reduced in size in the mutant larvae and fail to recover to control size at subsequent stages. In contrast, rods form temporarily expanded outer segments. The inner segments, which generate the required energy and proteins for the outer segments, are shortened in both rods and cones at all stages. RNA-Seq analysis provides a set of misregulated genes associated with the observed abnormal photoreceptor morphogenesis. Conclusions: GDF6 mutations were previously identified in patients with Leber congenital amaurosis. Here, we reveal a unique photoreceptor phenotype in the gdf6a mutant zebrafish whereby rods and cones undergo abnormal maturation distinct for each cell type. Further, subsequent development shows partial recovery of cell morphology and maintenance of the photoreceptor layer. By conducting a transcriptomic analysis of the gdf6a larval eyes, we identified a collection of genes that are candidate regulators of photoreceptor size and morphology.

Visualization of the Superior Ocular Sulcus during Danio rerio Embryogenesis.

Congenital ocular coloboma is a genetic disorder that is typically observed as a cleft in the inferior aspect of the eye resulting from incomplete choroid fissure closure. Recently, the identification of individuals with coloboma in the superior aspect of the iris, retina, and lens led to the discovery of a novel structure, referred to as the superior fissure or superior ocular sulcus (SOS), that is transiently present on the dorsal aspect of the optic cup during vertebrate eye development. Although this structure is conserved across mice, chick, fish, and newt, our current understanding of the SOS is limited. In order to elucidate factors that contribute to its formation and closure, it is imperative to be able to observe it and identify abnormalities, such as delay in the closure of the SOS. Here, we set out to create a standardized series of protocols that can be used to efficiently visualize the SOS by combining widely available microscopy techniques with common molecular biology techniques such as immunofluorescent staining and mRNA overexpression. While this set of protocols focuses on the ability to observe SOS closure delay, it is adaptable to the experimenter's needs and can be easily modified. Overall, we hope to create an approachable method through which our understanding of the SOS can be advanced to expand the current knowledge of vertebrate eye development.

Targeting of retinal axons requires the metalloproteinase ADAM10.

The role of extrinsic cues in guiding developing axons is well established; however, the means by which the activity of these extrinsic cues is regulated is poorly understood. A disintegrin and metalloproteinase (ADAM) enzymes are Zn-dependent proteinases that can cleave guidance cues or their receptors in vitro. Here, we identify the first example of a metalloproteinase that functions in vertebrate axon guidance in vivo. Specifically, ADAM10 is required for formation of the optic projection by Xenopus retinal ganglion cell (RGC) axons. Xadam10 mRNA is expressed in the dorsal neuroepithelium through which RGC axons extend. Pharmacological or molecular inhibition of ADAM10 within the brain each resulted in a failure of RGC axons to recognize their target. In contrast, molecular inhibition of ADAM10 within the RGC axons themselves had no effect. These data argue strongly that in the dorsal brain ADAM10 acts cell non-autonomously to regulate the guidance of RGC axons.