Shin-Ichi Higashijima

The acquisition of paired appendages was a key innovation in vertebrate evolution. Although HoxA/D paralog groups (PGs) 9–13 play central roles in patterning paired fins and limbs, the evolutionary origin of this paired appendage-patterning system remains unknown. Here, we show that HoxA/D PG9–10 genes are essential for the early establishment of pelvic fin progenitors in zebrafish, and that Hox9–10 function is similarly required for hindlimb progenitor formation in the Iberian ribbed newt. In zebrafish, combined disruption of five HoxA/D PG9–10 genes abolishes the detectable pelvic fin progenitor population, resulting in complete loss of pelvic fins. A Tg(hoxa10b:EGFP) reporter labeled cells in the presumptive pelvic fin field before pelvic fin bud formation. Importantly, simultaneous CRISPR-mediated disruption of all seven Hox9–10 paralogs in the Iberian ribbed newt similarly impaired the establishment of hindlimb progenitors, suggesting that an early requirement for Hox9–10 function in hind appendage development predates the divergence of ray-finned and lobe-finned vertebrates. Together, these findings identify an evolutionarily conserved role for posterior Hox9–10 genes in the initial establishment of hind appendage progenitors. Because overlapping posterior Hox paralog groups are subsequently deployed during paired appendage patterning, this early Hox9–10 requirement provides a developmental link between the posterior axial Hox code and the paired appendage-patterning system. Our results are therefore consistent with the hypothesis that recruitment of a posterior Hox code into hind appendage progenitor formation contributed to the evolutionary assembly of the vertebrate paired appendage-patterning system.

Oxytocin is a key regulator of social behavior, yet how it shapes the neural circuits underlying these behaviors remains unclear. Here, we show that oxytocin signaling is required for the proper maturation of terminal nerve gonadotropin-releasing hormone 3 (TN-GnRH3) circuits that regulate female mate preference in medaka. Disruption of oxytocin signaling reduced expression of the complement component C1q, altered microglial properties, and increased TN-GnRH3 innervation in the optic tectum and dorsolateral telencephalon, suggesting impaired synaptic refinement. In addition, TN-GnRH3 neurons in mutants exhibited disrupted clustering and failed to increase firing frequency in response to visual stimulation, indicating functional deficits. Furthermore, c1qb mutants phenocopied the behavioral and neuronal abnormalities observed in oxytocin signaling–deficient mutants. Together, our findings suggest that oxytocin signaling links microglia-associated synaptic refinement to neural circuit maturation, thereby revealing a previously unrecognized role for oxytocin in shaping social behavior.

Animals stabilize their heads to ensure stable sensory input and effective motor coordination. Head stabilization in response to vestibular stimuli is mediated by the vestibulo-collic reflex (VCR). While the VCR has been characterized in tetrapods, it remains unknown whether fish, which lack an anatomical neck, employ head-stabilization behavior. Here, we demonstrate that larval zebrafish exhibit head-stabilization behavior: during pitch tilts, they adjust their head orientation relative to the body by rostral body flexion. The rostral body flexes ventrally during head-up posture, whereas it flexes dorsally during head-down posture. These flexions partially compensate for head pitch changes, contributing to head stabilization. We identify the neural circuits and muscles responsible for these flexions. Both dorsal and ventral flexions are mediated by the same vestibular nucleus, but neural signals were transmitted through distinct pathways, either involving or bypassing a class of reticulospinal neurons. The dorsal and ventral flexions are produced by specialized dorsal and ventral muscles in the rostral body, respectively. The neural circuits underlying these body flexions share similarities with those underlying the mammalian VCR. Together, our results suggest that a head-stabilization system may have existed in ancestral fish and may have provided an evolutionary foundation upon which the tetrapod VCR emerged.

Head stabilization is essential for animal survival, enabling stable sensory input and effective motor coordination. Animals stabilize their heads in response to vestibular stimuli through the vestibulo-collic reflex (VCR). While the VCR has been characterized in tetrapod vertebrates, it remains unknown whether fish, which lack an anatomical neck, employ a comparable behavior. Here, we demonstrate that larval zebrafish exhibit VCR-like behaviors: they adjust their head orientation relative to the body by rostral body flexion during pitch tilts. The rostral body flexed ventrally during head-up posture, whereas it flexed dorsally during head-down posture. These flexions partially compensated for the head pitch changes, thereby contributing to head stabilization. We also identified the muscles and neural circuits responsible for these two types of body flexions. Both the dorsal and ventral flexions were mediated by the same vestibular nucleus, but neural signals were transmitted through distinct pathways, either involving or bypassing a class of reticulospinal neurons. The dorsal and ventral flexions were ultimately produced by specialized dorsal and ventral muscles in the rostral body, respectively. The neural circuits underlying these body flexions in fish share similarities with those underlying the mammalian VCR. Together, our results demonstrate that fish exhibit a VCR-like behavior through comparable circuit mechanisms, suggesting that the VCR is evolutionarily conserved across vertebrates.

Summary: Posture control is crucial for animals. Static posture control in fish remains poorly explored. Recent studies have shown that larval zebrafish perform body flexion during slight roll tilts: the body flexion displaces a gas-filled swim bladder, generating counter-rotation torque for postural recovery through misalignment of gravity and buoyancy forces. Swim bladder deflation impairs this postural recovery, suggesting its critical role. This static posture control strategy may be utilized by many fish species. However, adult fish differ from larvae in morphology and behavior, raising questions about the generality of this mechanism. Our behavioral analysis showed that adult zebrafish also flexed their body during roll tilts, with flexion persisting until recovery to an upright posture. Similarly to larvae, swim bladder deflation impaired postural recovery. These results demonstrate that adult fish employ static roll posture control through body flexion and suggest the generality of this mechanism in fish species.

In vertebrates, skeletal muscle comprises fast and slow fibers. Slow and fast muscle cells in fish are spatially segregated; slow muscle cells are located only in a superficial region, and comprise a small fraction of the total muscle cell mass. Slow muscles support low-speed, low-force movements, while fast muscles are responsible for high-speed, high-force movements. However, speed and strength of movement are not binary states, but rather fall on a continuum. This raises the question of whether any recruitment patterns exist within fast muscles, which constitute the majority of muscle cell mass. In the present study, we investigated activation patterns of trunk fast muscles during movements of varying speeds and strengths using larval zebrafish. We employed two complementary methods: calcium imaging and electrophysiology. The results obtained from both methods supported the conclusion that there are spatially-ordered recruitment patterns in fast muscle cells. During weaker/slower movements, only the lateral portion of fast muscle cells is recruited. As the speed or strength of the movements increases, more fast muscle cells are recruited in a spatially-ordered manner, progressively from lateral to medial. We also conducted anatomical studies to examine muscle fiber size. The results of those experiments indicated that muscle fiber size increases systematically from lateral to medial. Therefore, the spatially ordered recruitment of fast muscle fibers, progressing from lateral to medial, correlates with an increase in fiber size. These findings provide significant insights into the organization and function of fast muscles in larval zebrafish, illustrating how spatial recruitment and fiber size interact to optimize movement performance.

Patients with tauopathies present with profoundly different clinical symptoms ¹ , even within the same disorder ² . A central hypothesis in the field, well-supported by biomarker studies ^3,4 and post-mortem pathology ^5–7 , is that clinical heterogeneity reflects differential degeneration of vulnerable neuronal populations responsible for specific neurological functions. Recent work has revealed mechanisms underlying susceptibility of particular cell types ^8–10 , but relating tau load to disrupted behavior — es- pecially before cell death — requires a targeted circuit-level approach. Here we studied two distinct balance behaviors in larval zebrafish ¹¹ expressing a human 0N/4R-tau allele ¹² in select populations of evolutionarily-conserved and well-characterized brainstem vestibular circuits ^13,14 . We observed that human tau load predicted the severity of circuit-specific deficits in posture and navigation in the ab- sence of cell death. Targeting expression to either mid- or hindbrain balance neurons recapitulated these particular deficits in posture and navigation. By parametrically linking tau load in specific neu- rons to early behavioral deficits, our work moves beyond cell type to close the gap between pathological and neurological conceptions of tauopathy.

Mature vertebrates maintain posture using vestibulospinal neurons that transform sensed instability into reflexive commands to spinal motor circuits. Postural stability improves across development. However, due to the complexity of terrestrial locomotion, vestibulospinal contributions to postural refinement in early life remain unexplored. Here we leveraged the relative simplicity of underwater locomotion to quantify the postural consequences of losing vestibulospinal neurons during development in larval zebrafish of undifferentiated sex. By comparing posture at two timepoints, we discovered that later lesions of vestibulospinal neurons led to greater instability. Analysis of thousands of individual swim bouts revealed that lesions disrupted movement timing and corrective reflexes without impacting swim kinematics, and that this effect was particularly strong in older larvae. Using a generative model of swimming, we showed how these disruptions could account for the increased postural variability at both timepoints. Finally, late lesions disrupted the fin/trunk coordination observed in older larvae, linking vestibulospinal neurons to postural control schemes used to navigate in depth. Since later lesions were considerably more disruptive to postural stability, we conclude that vestibulospinal contributions to balance increase as larvae mature. Vestibulospinal neurons are highly conserved across vertebrates; we therefore propose that they are a substrate for developmental improvements to postural control.

Mature vertebrates maintain posture using vestibulospinal neurons that transform sensed instability into reflexive commands to spinal motor circuits. Postural stability improves across development. However, due to the complexity of terrestrial locomotion, vestibulospinal contributions to postural refinement in early life remain unexplored. Here we leveraged the relative simplicity of underwater locomotion to quantify the postural consequences of losing vestibulospinal neurons during development in larval zebrafish of undifferentiated sex. By comparing posture at two timepoints, we discovered that later lesions of vestibulospinal neurons led to greater instability. Analysis of thousands of individual swim bouts revealed that lesions disrupted movement timing and corrective reflexes without impacting swim kinematics, and that this effect was particularly strong in older larvae. Using a generative model of swimming, we showed how these disruptions could account for the increased postural variability at both timepoints. Finally, late lesions disrupted the fin/trunk coordination observed in older larvae, linking vestibulospinal neurons to postural control schemes used to navigate in depth. Since later lesions were considerably more disruptive to postural stability, we conclude that vestibulospinal contributions to balance increase as larvae mature. Vestibulospinal neurons are highly conserved across vertebrates; we therefore propose that they are a substrate for developmental improvements to postural control. SIGNIFICANCE STATEMENT: Many animals experience balance improvements during early life. Mature vertebrates use vestibulospinal neurons to transform sensed instability into postural corrections. To understand if/how these neurons shape postural development, we ablated them at two developmentally important timepoints in larval zebrafish. Loss of vestibulospinal neurons disrupted specific stabilizing behaviors (swim timing, tilt correction, and fin/body coordination) more profoundly in older fish. We conclude that postural development happens in part by changes to vestibulospinal neurons — a significant step towards understanding how developing brains gain the ability to balance.