The Fraser complex interconnects tissue layers to support basal epidermis and osteoblast integrated morphogenesis underlying fin skeletal patterning.

Fraser Syndrome is a rare, multisystemic autosomal recessive disorder characterized by disrupted epithelial-mesenchymal associations upon loss of Fraser Complex genes. Disease manifestation and affected organs are highly variable. Digit malformations such as syndactyly are common but of unclear developmental origins. We explored if zebrafish fraser extracellular matrix complex subunit 1 (fras1) mutants model Fraser Syndrome-associated appendicular skeleton patterning defects. Approximately 10% of fras1 mutants survive to adulthood, displaying striking and varied fin abnormalities, including endochondral bone fusions, ectopic cartilage, and disrupted caudal fin symmetry. The fins of surviving fras1 mutants frequently have fewer and unbranched bony rays. fras1 mutant fins regenerate to their original size but with exacerbated ray branching and fin symmetry defects. Single cell RNA-Seq analysis, in situ hybridizations, and antibody staining show specific Fraser complex expression in the basal epidermis during regenerative outgrowth. Fras1 and Fraser Complex component Frem2 accumulate along the basal side of distal-most basal epidermal cells. Greatly reduced and mislocalized Frem2 accompanies loss of Fras1 in fras1 mutants. The Sonic hedgehog signaling between distal basal epidermis and adjacent mesenchymal pre-osteoblasts that promotes ray branching persists upon Fraser Complex loss. However, fras1 mutant regenerating fins exhibit extensive sub-epidermal blistering associated with a disorganized basal epidermis and adjacent pre-osteoblasts. We propose Fraser Complex-supported tissue layer adhesion enables robust integrated tissue morphogenesis involving the basal epidermis and osteoblasts. Further, we establish zebrafish fin development and regeneration as an accessible model to explore mechanisms of Fraser Syndrome-associated digit defects and Fraser Complex function at epithelial-mesenchymal interfaces.

Insulin-like growth factor receptor / mTOR signaling elevates global translation to accelerate zebrafish fin regenerative outgrowth.

Zebrafish robustly regenerate fins, including their characteristic bony ray skeleton. Amputation activates intra-ray fibroblasts and dedifferentiates osteoblasts that migrate under a wound epidermis to establish an organized blastema. Coordinated proliferation and re-differentiation across lineages then sustains progressive outgrowth. We generate a single cell transcriptome dataset to characterize regenerative outgrowth and explore coordinated cell behaviors. We computationally identify sub-clusters representing most regenerative fin cell lineages, and define markers of osteoblasts, intra- and inter-ray fibroblasts and growth-promoting distal blastema cells. A pseudotemporal trajectory and in vivo photoconvertible lineage tracing indicate distal blastemal mesenchyme restores both intra- and inter-ray fibroblasts. Gene expression profiles across this trajectory suggest elevated protein production in the blastemal mesenchyme state. O-propargyl-puromycin incorporation and small molecule inhibition identify insulin growth factor receptor (IGFR)/mechanistic target of rapamycin kinase (mTOR)-dependent elevated bulk translation in blastemal mesenchyme and differentiating osteoblasts. We test candidate cooperating differentiation factors identified from the osteoblast trajectory, finding IGFR/mTOR signaling expedites glucocorticoid-promoted osteoblast differentiation in vitro. Concordantly, mTOR inhibition slows but does not prevent fin regenerative outgrowth in vivo. IGFR/mTOR may elevate translation in both fibroblast- and osteoblast-lineage cells during the outgrowth phase as a tempo-coordinating rheostat.

Coordinated patterning of zebrafish caudal fin symmetry by a central and two peripheral organizers.

BACKGROUND: Caudal fin symmetry characterizes teleosts and likely contributes to their evolutionary success. However, the coordinated development and patterning of skeletal elements establishing external symmetry remains incompletely understood. We explore the spatiotemporal emergence of caudal skeletal elements in zebrafish to consider evolutionary and developmental origins of caudal fin symmetry. RESULTS: Transgenic reporters and skeletal staining reveal that the hypural diastema-defining gap between hypurals 2 and 3 forms early and separates progenitors of two plates of connective tissue. Two sets of central principal rays (CPRs) synchronously, sequentially, and symmetrically emerge around the diastema. The two dorsal- and ventral-most rays (peripheral principal rays, PPRs) arise independently and earlier than adjacent CPRs. Muscle and tendon markers reveal that different muscles attach to CPR and PPR sets. CONCLUSIONS: We propose that caudal fin symmetry originates from a central organizer that establishes the hypural diastema and bidirectionally patterns surrounding tissue into two plates of connective tissue and two mirrored sets of CPRs. Further, two peripheral organizers unidirectionally specify PPRs, forming a symmetric "composite" fin derived from three fields. Distinct CPR and PPR ontogenies may represent developmental modules conferring ray identities, muscle connections, and biomechanical properties. Our model contextualizes mechanistic studies of teleost fin morphological variation.

Extensively Drug-Resistant Typhoid Fever in the United States.

Cases of extensively drug-resistant (XDR) typhoid fever have been reported in the United States among patients who did not travel internationally. Clinicians should consider if and where the patient traveled when selecting empiric treatment for typhoid fever. XDR typhoid fever should be treated with a carbapenem, azithromycin, or both.

longfin causes cis-ectopic expression of the kcnh2a ether-a-go-go K+ channel to autonomously prolong fin outgrowth.

Organs stop growing to achieve a characteristic size and shape in scale with the body of an animal. Likewise, regenerating organs sense injury extents to instruct appropriate replacement growth. Fish fins exemplify both phenomena through their tremendous diversity of form and remarkably robust regeneration. The classic zebrafish mutant longfint2 develops and regenerates dramatically elongated fins and underlying ray skeleton. We show longfint2 chromosome 2 overexpresses the ether-a-go-go-related voltage-gated potassium channel kcnh2a. Genetic disruption of kcnh2a in cis rescues longfint2, indicating longfint2 is a regulatory kcnh2a allele. We find longfint2 fin overgrowth originates from prolonged outgrowth periods by showing Kcnh2a chemical inhibition during late stage regeneration fully suppresses overgrowth. Cell transplantations demonstrate longfint2-ectopic kcnh2a acts tissue autonomously within the fin intra-ray mesenchymal lineage. Temporal inhibition of the Ca2+-dependent phosphatase calcineurin indicates it likewise entirely acts late in regeneration to attenuate fin outgrowth. Epistasis experiments suggest longfint2-expressed Kcnh2a inhibits calcineurin output to supersede growth cessation signals. We conclude ion signaling within the growth-determining mesenchyme lineage controls fin size by tuning outgrowth periods rather than altering positional information or cell-level growth potency.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins.

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

Cross-sex testosterone therapy in ovariectomized mice: addition of low-dose estrogen preserves bone architecture.

Cross-sex hormone therapy (XHT) is widely used by transgender people to alter secondary sex characteristics to match their desired gender presentation. Here, we investigate the long-term effects of XHT on bone health using a murine model. Female mice underwent ovariectomy at either 6 or 10 wk and began weekly testosterone or vehicle injections. Dual-energy X-ray absorptiometry (DXA) was performed (20 wk) to measure bone mineral density (BMD), and microcomputed tomography was performed to compare femoral cortical and trabecular bone architecture. The 6-wk testosterone group had comparable BMD with controls by DXA but reduced bone volume fraction, trabecular number, and cortical area fraction and increased trabecular separation by microcomputed tomography. Ten-week ovariectomy/XHT maintained microarchitecture, suggesting that estrogen is critical for bone acquisition during adolescence and that late, but not early, estrogen loss can be sufficiently replaced by testosterone alone. Given these findings, we then compared effects of testosterone with effects of weekly estrogen or combined testosterone/low-dose estrogen treatment after a 6-wk ovariectomy. Estrogen treatment increased spine BMD and microarchitecture, including bone volume fraction, trabecular number, trabecular thickness, and connectivity density, and decreased trabecular separation. Combined testosterone-estrogen therapy caused similar increases in femur and spine BMD and improved architecture (increased bone volume fraction, trabecular number, trabecular thickness, and connectivity density) to estrogen therapy and were superior compared with mice treated with testosterone only. These results demonstrate estradiol is critical for bone acquisition and suggest a new cross-sex hormone therapy adding estrogens to testosterone treatments with potential future clinical implications for treating transgender youth or men with estrogen deficiency.

Six-year retrospective study of reconstructive options for defects of the skull base after resection of tumour.

The successful management of tumours that involve the skull base depends on resection and reconstruction of the defect. The challenge is to create a protective vascularised seal between the intracranial contents and the adjacent paranasal sinuses, nasopharynx, and oral cavity. Failure to create a protective barrier for the intracranial contents can result in cerebrospinal fistulas, potentially fatal infections, and radiotherapy being delayed. Local or regional flaps may be adequate for small defects, but larger ones require microsurgical free tissue transfer. As a part of the skull base surgical team, we present our experience in the reconstruction of defects after resection of tumours. A total of 41 patients had operations over a 6-year period from January 2004 to January 2010, and 26 free tissue transfer flaps and 20 local or regional flaps (46 flaps) were used for reconstruction. Ninety-two percent of the free flaps were successful. We also report details on disease, complications, and survival at 3, 6, and 12 months. Survival depended not only on the type of disease, coexisting conditions, and adequate resection of tumour, but equally on the reconstruction of the defect.