Postembryonic development of the posterior lateral line in the zebrafish

SUMMARY The posterior lateral line (PLL) of zebrafish comprises seven to eight sense organs at the end of embryogenesis, arranged in a single antero-posterior line that extends along the horizontal myoseptum from the ear to the tip of the tail. At the end of larval life, four antero-posterior lines extend on the trunk and tail, comprising together around 60 sense organs. The embryonic pattern is largely conserved among teleosts, although adult patterns are very diverse. Here we describe the transition from embryonic to juvenile pattern in the zebrafish, to provide a framework for understanding how the diversity of adult patterns comes about. We show that the four lines that extend over the adult body originate from latent precursors laid down by migrating primordia that arise during embryogenesis. We conclude that, in zebrafish, the entire development of the PLL system up to adulthood can be traced back to events that took place during the first 2 days of life. We also show that the transition from embryonic to adult pattern involves few distinct operations, suggesting that the diversity of patterns among adult teleosts may be due to differential control of these few operations acting upon common embryonic precursors.     

Partial Absence of Pleuropericardial Membranes in Tbx18- and Wt1-Deficient Mice

The pleuropericardial membranes are fibro-serous walls that separate the pericardial and pleural cavities and anchor the heart inside the mediastinum. Partial or complete absence of pleuropericardial membranes is a rare human disease, the etiology of which is poorly understood. As an attempt to better understand these defects, we wished to analyze the cellular and molecular mechanisms directing the separation of pericardial and pleural cavities by pleuropericardial membranes in the mouse. We found by histological analyses that both in Tbx18- and Wt1-deficient mice the pleural and pericardial cavities communicate due to a partial absence of the pleuropericardial membranes in the hilus region. We trace these defects to a persisting embryonic connection between these cavities, the pericardioperitoneal canals. Furthermore, we identify mesenchymal ridges in the sinus venosus region that tether the growing pleuropericardial membranes to the hilus of the lung, and thus, close the pericardioperitoneal canals. In Tbx18-deficient embryos these mesenchymal ridges are not established, whereas in Wt1-deficient embryos the final fusion process between these tissues and the body wall does not occur. We suggest that this fusion is an active rather than a passive process, and discuss the interrelation between closure of the pericardioperitoneal canals, lateral release of the pleuropericardial membranes from the lateral body wall, and sinus horn development.     

The overlapping roles of the inner ear and lateral line: the active space of dipole source detection

The problems associated with the detection of sounds and other mechanical disturbances in the aquatic environment differ greatly from those associated with airborne sounds. The differences are primarily due to the incompressibility of water and the corresponding increase in importance of the acoustic near field. The near field, or hydrodynamic field, is characterized by steep spatial gradients in pressure, and detection of the accelerations associated with these gradients is performed by both the inner ear and the lateral line systems of fishes. Acceleration-sensitive otolithic organs are present in all fishes and provide these animals with a form of inertial audition. The detection of pressure gradients, by both the lateral line and inner ear, is the taxonomically most widespread mechanism of sound-source detection amongst vertebrates, and is thus the most likely primitive mode of detecting sound sources. Surprisingly, little is known about the capabilities of either the lateral line or the otolithic endorgan in the detection of vibratory dipole sources. Theoretical considerations for the overlapping roles of the inner ear and lateral line systems in midwater predict that the lateral line will operate over a shorter distance range than the inner ear, although with a much greater spatial resolution. Our empirical results of dipole detection by mottled sculpin, a benthic fish, do not agree with theoretical predictions based on midwater fishes, in that the distance ranges of the two systems appear to be approximately equal. This is almost certainly as a result of physical coupling between the fishes and the substrate. Thus, rather than having a greater active range, the inner ear appears to have a reduced distance range in benthic fishes, and the lateral line distance range may be concomitantly extended.     

Cells of origin of the branches of the facial nerve: a retrograde HRP study in the rabbit

The origin of different branches of the facial nerve in the rabbit was determined by using retrograde transport of HRP. Either the proximal stump of specific nerves was exposed to HRP after transection, or an injection of the tracer was made into particular muscles innervated by a branch of the facial nerve. A clear somatotopic pattern was observed. Those branches which innervate the rostral facial musculature arise from cells located in the lateral and intermediate portions of the nuclear complex. Orbital musculature is supplied by neurons in the dorsal portion of the complex, with the more rostral orbital muscles receiving input from more laterally located cells while the caudal orbital region receives innervation from more medial regions of the dorsal facial nucleus. The rostral portion of the ear also receives innervation from cells located in the dorsomedial part of the nucleus, but the caudal aspect of the ear is supplied exclusively by cells located in medial regions. The cervical platysma, the platysma of the lower jaw, and the deep muscles (i.e., digastric and stylohyoid) receive input from cells topographically arranged in the middle and ventral portions of the nuclear complex. It is proposed that the topographic relationship between the facial nucleus and branches of the facial nerve reflects the embryological derivation of the facial muscles. Those muscles that develop from the embryonic sphincter colli profundus layer are innervated by lateral and dorsomedial portions of the nuclear complex. The muscles derived from the embryonic platysma layer, including the deep musculature, receive their input from mid to ventral regions of the nuclear complex.     

Functional anatomy of the canine mediastinal cardiac nerves located at the base of the heart

The major canine cardiopulmonary nerves which arise from the middle cervical and stellate ganglia and the vagi course toward the heart in the dorsal mediastinum where they form, at the base of the heart dorsal to the pulmonary artery and aorta, the dorsal mediastinal cardiac nerves. In addition, the left caudal pole and interganglionic nerves project onto the left lateral side of the heart as the left lateral cardiac nerve. These nerves contain afferent and (or) efferent axons which, upon stimulation, modify specific cardiac regions and (or) systemic pressure. In addition, with the exception of the left lateral cardiac nerve, stimulation of each of these nerves produces compound action potentials in the cranial ends of the majority of the major cardiopulmonary nerves demonstrating that axons in each dorsal mediastinal cardiac nerve interconnect with axons in the majority of the cardiopulmonary nerves. Axons in the left lateral cardiac nerve connect primarily with axons in the left caudal pole and left interganglionic nerves. The dorsal mediastinal nerves project distally onto the heart as coronary nerves accompanying the right or left coronary arteries. These innervated the ventricular myocardium which is supplied by their respective vessels. The left lateral cardiac nerve projects directly onto the lateral epicardium of the left ventricle. The dorsal mediastinal and left lateral cardiac nerves are the major sympathetic cardiac nerves. Thus, the cardiac nerves located in the mediastinum at the base of the heart are not simple extensions of cardiopulmonary nerves, but rather have a unique anatomy and function of their own.     

Shaping sound in space: the regulation of inner ear patterning

The inner ear is one of the most morphologically elaborate tissues in vertebrates, containing a group of mechanosensitive sensory organs that mediate hearing and balance. These organs are arranged precisely in space and contain intricately patterned sensory epithelia. Here, we review recent studies of inner ear development and patterning which reveal that multiple stages of ear development - ranging from its early induction from the embryonic ectoderm to the establishment of the three cardinal axes and the fine-grained arrangement of sensory cells - are orchestrated by gradients of signaling molecules.     

Development of the Vascular System in the Ear of Barley

In the mature rachis internodes of a two-row barley ear therewere two large lateral vascular bundles and a number of smallermedian and outer lateral vascular bundles. These bundles rannearly the full length of the rachis with no cross connectionsbetween them. The lateral and the outer lateral bundles branchedat every node while the median bundles branched at every othernode. The vascular bundles to each spikelet passed through acomplex region in which transfer cells occurred. Development of the vascular system of the young main shoot earstarted at the triple mound stage when the procambium of lateralbundles was initiated. Initiation of the median bundles andthe outer lateral bundles followed in sequence with some differentiationof protophloem and protoxylem. At awn initial stage of the earthe vascular connections to the spikelets were established withdifferentiation of the almost complete procambium proceedingthroughout the ear elongation phase. The progress of vascularisation in the young ear is discussedwith reference to spikelet initiation, ear relative growth rateand death of terminal spikelets. It is proposed that the changesin the relative growth rate of the ear and death of the terminalspikelets may be related to vascular differentiation     

Development of the vascular bed in the rabbit ear: scanning electron microscopy of vascular corrosion casts

The development of the vascular bed in the rabbit ear was investigated using vascular corrosion casts from animals of various ages. Examination of the casts revealed that the arrangement of the major auricular arteries and veins was determined before birth and was maintained during postnatal growth of the ear. Furthermore, the number of arteries branching off the central ear artery and the lateral arteries did not increase with increasing ear length. Scanning electron microscopic examination of lateral segments of adult ear casts revealed many anastomoses between marginal arteries and veins. These arteriovenous anastomoses (AVAs) occurred singly, in pairs, or in clusters of three to six. Their size and shape were variable, even in the same cast. The central segment of many AVA casts showed surface impressions of endothelial cell nuclei which were different from the impressions on adjacent arteries and veins. Arteriovenous anastomoses were also detected in ear casts from animals as young as 8 days. The density of AVAs in lateral ear segments ranged from 95-165 cm-2 in 8- to 11-day-old rabbits to 80-115 cm-2 in adults. However, estimates of the total number of AVAs in the lateral ear margin indicated that AVAs continued to be formed at a steady rate during growth of the ear. During the early neonatal period the cutaneous capillary plexuses developed prominent tufts projecting toward the skin surface, which were apparently associated with developing hair follicles. These capillary tufts were not seen in casts from fetal or adult rabbits.     

Middle-ear development: II. Morphometric changes in the conducting apparatus of the chick.

The ontogeny of various middle-ear structures was examined in 11 groups of chicks between 10 days embryonic and adult. Measurements of the tympanic membrane surface area and height, columella length, and that of the columella footplate, annular ligament, and oval window area were obtained using video micrographs and computer digitization techniques. The oval window matures first at 53 days post-hatching, whereas the columella achieves adult size at 74 days. The tympanic membrane surface area is the last middle-ear variable studied to reach adult size (79 days post-hatch). The columella increases its length from 0.63 mm (10 days embryonic) to 2.73 mm in the adult. The tympanic membrane area expands by 280% whereas the columellar footplate area increases by 11x. As a result, the pressure amplification of the middle ear due to the tympanic membrane/columellar footplate area ratio improves by over 400%. These data further contribute to our understanding of the functional development of the middle ear.     

In vitro development of the embryonic mouse inner ear following exposure to trypsin

Trypsin has been shown to disrupt normal in vitro morphogenesis of embryonic organ rudiments. Otic tissues derived from 11-, 12-, and 13-day-old mouse embryos were exposed to either Ca++- and Mg++-free PBS or 0.25% trypsin dissolved in Ca++- and Mg++-free PBS prior to explanation into organ culture. Trypsin treatment of otic explants disrupted the expression of the normal pattern of inner-ear development in vitro. There was a direct correlation between the embryonic age at time of exposure to trypsin and the severity of dysmorphogenesis of the inner ear. The younger explants showed abnormalities of both vestibular and auditory structures, whereas with increasing embryonic age, abnormalities were confined more to the auditory portion of the inner ear. The results suggest that integrity of the otocyst basal lamina and epitheliomesenchymal tissue interactions are important factors in early otic development. It is postulated that the major effect of trypsin on inner-ear morphogenesis is through disruption of these factors, which may act to regulate the progressive expression of early otic development.     

Lateral line, otic and epibranchial placodes: developmental and evolutionary links?

Two embryonic cell populations, the neural crest and cranial ectodermal placodes, between them give rise to many of the unique characters of vertebrates. Neurogenic placode derivatives are vital for sensing both external and internal stimuli. In this speculative review, we discuss potential developmental and evolutionary relationships between two placode series that are usually considered to be entirely independent: lateral line placodes, which form the mechanosensory and electroreceptive hair cells of the anamniote lateral line system as well as their afferent neurons, and epibranchial placodes (geniculate, petrosal and nodose), which form Phox2b(+) visceral sensory neurons with input from both the external and internal environment. We illustrate their development using molecular data we recently obtained in shark embryos, and we describe their derivatives, including the possible geniculate placode origin of a mechanosensory sense organ associated with the first pharyngeal pouch/cleft (the anamniote spiracular organ/amniote paratympanic organ). We discuss how both lateral line and epibranchial placodes can be related in different ways to the otic placode (which forms the inner ear and its afferent neurons), and how both are important for protective somatic reflexes. Finally, we put forward a highly speculative proposal about the original function of the cells whose evolutionary descendants today include the derivatives of the lateral line, otic and epibranchial placodes, namely that they produced sensory receptors and neurons for Phox2b-dependent protective reflex circuits. We hope this review will stimulate both debate and a fresh look at possible developmental and evolutionary relationships between these seemingly disparate and independent placodes.     

The Wheels mutation in the mouse causes vascular, hindbrain, and inner ear defects

In a screen for mouse mutations with dominant behavioral anomalies, we identified Wheels, a mutation associated with circling and hyperactivity in heterozygotes and embryonic lethality in homozygotes. Mutant Wheels embryos die at E10.5-E11.5 and exhibit a host of morphological anomalies which include growth retardation and anomalies in vascular and hindbrain development. The latter includes perturbation of rhombomeric boundaries as detected by Krox20 and Hoxb1. PECAM-1 staining of embryos revealed normal formation of the primary vascular plexus. However, subsequent stages of branching and remodeling do not proceed normally in the yolk sac and in the embryo proper. To obtain insights into the circling behavior, we examined development of the inner ear by paint-filling of membranous labyrinths of Whl/+ embryos. This analysis revealed smaller posterior and lateral semicircular canal primordia and a delay in the canal fusion process at E12.5. By E13.5, the lateral canal was truncated and the posterior canal was small or absent altogether. Marker analysis revealed an early molecular phenotype in heterozygous embryos characterized by perturbed expression of Bmp4 and Msx1 in prospective lateral and posterior cristae at E11.5. We have constructed a genetic and radiation hybrid map of the centromeric portion of mouse Chromosome 4 across the Wheels region and refined the position of the Wheels locus to the approximately 1.1-cM region between D4Mit104 and D4Mit181. We have placed the locus encoding Epha7, in the Wheels candidate region; however, further analysis showed no mutations in the Epha7-coding region and no detectable changes in mRNA expression pattern. In summary, our findings indicate that Wheels, a gene which is essential for the survival of the embryo, may link diverse processes involved in vascular, hindbrain, and inner ear development.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Immunocytochemical study of the development of vasotocin/mesotocin in the hypothalamo-hypophysial system of the chick embryo

Summary The hypothalamo-hypophysial system of the chick embryo has been studied with a monoclonal antibody which cross-reacts with arginine vasotocin and mesotocin, using thick (100 m) sections in conjunction with a peroxidase-conjugated rabbit anti-mouse antibody. Although weakly stained perikarya occur occasionally in the tuberal region on embryonic days 6 and 7, the most consistent immunostaining of perikarya is found in the periventricular region of the caudal midhypothalamus at the level of the optic chiasm after embryonic day 8 1/2. Synthesis of peptides, therefore, takes place while the cells are close to their site of origin. Between embryonic days 9 and 10, beaded axons run along the anterior median eminence closely apposed to the adenohypophysis, thereby forming the anlage of the zona externa. The axons of the hypothalamo-neurohypophysial tract surround the neural lobe between embryonic days 11 and 12. The caudal to rostral wave of neuronal maturation that occurs during development appears to be due to a progressive differentiation of the periventricular zone, as well as the migration of perikarya. The early periventricular perikarya at embryonic day 8 1/2 send processes rostrally in a wing-shaped formation that extends both dorso- and ventrolaterally. From embryonic days 10 to 12, perikarya can be observed in the wing-like extensions, apparently migrating to rostral levels. The dorsolateral pathway gives rise at its midportion to the lateral cell group, whereas those perikarya migrating more laterally form the anlage of the external supraoptic nucleus. The ventrolateral wing-shaped extension of perikarya appears to be directed toward the ventral group and those lateral perikarya continuous with it. The location of mature neuronal cell groups is well established by embryonic day 17.     

Ionentransport und Taubheit

The identification of deafness genes helped to unravel the molecular mechanisms of ion movements that underlie the hearing process in the inner ear. Sound waves cause movements of the tympanic membrane that are transmitted as fluid movements to the inner ear by the middle ear bones. The sound-induced movements deflect hair cell stereocilia, which are bathed in endolymph. These movements cause the opening of mechanosensitive ion channels. Because of the high potassium concentration of the endolymph, potassium floods into the hair cells, which then depolarize. This results in transmitter release and the generation of postsynaptic electrical signals which are transmitted via the cochlear nerve. The unique ion gradient between hair cells and the endolymph is generated by a highly specialized epithelium in the lateral wall of the scala media, the stria vascularis.     

Building inner ears: recent advances and future challenges for in vitro organoid systems

While inner ear disorders are common, our ability to intervene and recover their sensory function is limited. In vitro models of the inner ear, like the organoid system, could aid in identifying new regenerative drugs and gene therapies. Here, we provide a perspective on the status of in vitro inner ear models and guidance on how to improve their applicability in translational research. We highlight the generation of inner ear cell types from pluripotent stem cells as a particularly promising focus of research. Several exciting recent studies have shown how the developmental signaling cues of embryonic and fetal development can be mimicked to differentiate stem cells into “inner ear organoids” containing otic progenitor cells, hair cells, and neurons. However, current differentiation protocols and our knowledge of embryonic and fetal inner ear development in general, have a bias toward the sensory epithelia of the inner ear. We propose that a more holistic view is needed to better model the inner ear in vitro. Moving forward, attention should be made to the broader diversity of neuroglial and mesenchymal cell types of the inner ear, and how they interact in space or time during development. With improved control of epithelial, neuroglial, and mesenchymal cell fate specification, inner ear organoids would have the ability to truly recapitulate neurosensory function and dysfunction. We conclude by discussing how single-cell atlases of the developing inner ear and technical innovations will be critical tools to advance inner ear organoid platforms for future pre-clinical applications.Subject terms: Cell biology, Somatic system, Stem-cell research