Dynamics of the suprapatellar bursa during knee joint extension.

PURPOSE: The suprapatellar bursa is located in the proximal deep layer of the patella and is thought to reduce tissue friction by changing from a single-membrane structure to a double-membrane structure during knee joint motion. However, the dynamics of the suprapatellar bursa have only been inferred from positional relationships, and the actual dynamics have not been confirmed. METHODS: Dynamics of the suprapatellar bursa during knee joint motion were observed in eight knees of four Thiel-fixed cadavers and the angle at which the bursa begins to show a double membrane was revealed. The flexion angles of knee joints were measured when the double-membrane structure of the suprapatellar bursa began to appear during knee joint extension. RESULTS: The suprapatellar bursa changes from a single membrane to a double-membrane structure at 91 ± 4° of flexion, when the knee joint is moved from a flexed position to an extended position. CONCLUSION: The suprapatellar bursa may be involved in limitations to knee joint range of motion and pain at an angle of approximately 90°. Further studies are needed to verify whether the same dynamics are observed in living subjects.

Development and validation of a machine learning model to predict time to renal replacement therapy in patients with chronic kidney disease.

BACKGROUND: Predicting time to renal replacement therapy (RRT) is important in patients at high risk for end-stage kidney disease. We developed and validated machine learning models for predicting the time to RRT and compared its accuracy with conventional prediction methods that uses the rate of estimated glomerular filtration rate (eGFR) decline. METHODS: Data of adult chronic kidney disease (CKD) patients who underwent hemodialysis at Oita University Hospital from April 2016 to March 2021 were extracted from electronic medical records (N = 135). A new machine learning predictor was compared with the established prediction method that uses the eGFR decline rate and the accuracy of the prediction models was determined using the coefficient of determination (R2). The data were preprocessed and split into training and validation datasets. We created multiple machine learning models using the training data and evaluated their accuracy using validation data. Furthermore, we predicted the time to RRT using a conventional prediction method that uses the eGFR decline rate for patients who had measured eGFR three or more times in two years and evaluated its accuracy. RESULTS: The least absolute shrinkage and selection operator regression model exhibited moderate accuracy with an R2 of 0.60. By contrast, the conventional prediction method was found to be extremely low with an R2 of -17.1. CONCLUSIONS: The significance of this study is that it shows that machine learning can predict time to RRT moderately well with continuous values from data at a single time point. This approach outperforms the conventional prediction method that uses eGFR time series data and presents new avenues for CKD treatment.

Spatiotemporal expression patterns of R-spondins and their receptors, Lgrs, in the developing mouse telencephalon.

Development of the mammalian telencephalon, which is the most complex region of the central nervous system, is precisely orchestrated by many signaling molecules. Wnt signaling derived from the cortical hem, a signaling center, is crucial for telencephalic development including cortical patterning and the induction of hippocampal development. Secreted protein R-spondin (Rspo) 1-4 and their receptors, leucine-rich repeat-containing G-protein-coupled receptor (Lgr) 4-6, act as activators of Wnt signaling. Although Rspo expression in the hem during the early stages of cortical development has been reported, comparative expression analysis of Rspos and Lgr4-6 has not been performed. In this study, we examined the detailed spatiotemporal expression patterns of Rspo1-4 and Lgr4-6 in the embryonic and postnatal telencephalon to elucidate their functions. In the embryonic day (E) 10.5-14.5 telencephalon, Rspo1-3 were prominently expressed in the cortical hem. Among their receptors, Lgr4 was observed in the ventral telencephalon, and Lgr6 was highly expressed throughout the telencephalon at the same stages. This suggests that Rspo1-3 and Lgr4 initially regulate telencephalic development in restricted regions, whereas Lgr6 functions broadly. From the late embryonic stage, the expression areas of Rspo1-3 and Lgr4-6 dramatically expanded; their expression was found in the neocortex and limbic system, such as the hippocampus, amygdala, and striatum. Increased Rspo and Lgr expression from the late embryonic stages suggests broad roles of Rspo signaling in telencephalic development. Furthermore, the Lgr+ regions were located far from the Rspo+ regions, especially in the E10.5-14.5 ventral telencephalon, suggesting that Lgrs act via a Rspo-independent pathway.

Kinetic study on heterogeneous nucleation and incubation period during chemical vapor deposition.

The kinetics of heterogeneous nucleation during chemical vapor deposition (CVD) is still unclear despite its importance. Nucleation delay is often observed in many CVD processes, which is known as the incubation period (τi). In this study, the effects of concentration (C) and sticking probability (η) of film-forming species on τi were formulated based on our kinetic model. To discuss the kinetics, τi -1 with the rate dimension was used and formulated using C and η. Because η onto heterogeneous surfaces (ηhetero) is difficult to evaluate, the study was initiated with η onto homogeneous surfaces (ηhomo), followed by a discussion on its reasonability. The formulation was validated using the experimental dataset for SiC-CVD from CH3SiCl3/H2 onto BN underlayers because CVD involves multiple film-forming species with different ηhomo ranging from 10-6 to 10-2 and thus is a suitable system for studying the effect of ηhomo. High-aspect-ratio (1000:1) parallel-plate microchannels consisting of τi-involving BN and a τi-free Si surface were utilized to separate these film-forming species along the microchannel depth. τi was exceptionally long, up to several hours, depending on the CVD conditions. τi -1 was found to be proportional to Cn, where n is the reaction order. n was quantified as ≈1.6, suggesting the initial nucleation was triggered by the impingement of two adspecies in the second order and lowered possibly by the discrepancy between C in the gas-phase and that actually producing adspecies on the surface. τi -1 was also found to be proportional to ηhomo. The exceptionally long τi was likely originated from the significantly lower ηhetero than ηhomo and the higher activation energy for ηhetero than that for ηhomo.

Macroscopic Anatomy of the Layered Structures of Facial Muscles and Fasciae in the Temporal-Malar-Mandible-Neck Region.

The layered structures of facial muscles and their topographical relationship with facial fasciae are still not fully understood. This study aimed to clarify the layered structures of facial muscles and fasciae in the temporal-malar-mandible-neck region. Thirty-four human cadavers were examined during gross anatomy courses at Niigata University (2017-2020). The face was composed of 3-layered (deep, middle, and superficial) fasciae and 4-layered facial muscles (first superficial, second superficial, third, and fourth muscle layers) according to the attachment of muscles and their topographical relationship with the fasciae. The deep fascia covered the temporal and masseter muscles. The parotid gland and facial nerves were enveloped in the middle fascia. The superficial fascia was continuous with the second superficial muscle layer. The connection between fourth and superficial muscles was at the malar and buccal areas, where the platysma blended with the masseter and the plural muscles blended with the buccinator. Our findings suggest that cooperation between the 4-layered structure of the facial muscles surrounding the apertures of the eyes and mouth and the superficial fascia enables humans to produce complex facial expressions. Furthermore, the spread of inflammation in the face may be owing to the layered facial muscles and fasciae, as these layered structures separate tissues into multiple compartments.

A three-component model of the spinal nerve ramification: Bringing together the human gross anatomy and modern Embryology.

Due to its long history, the study of human gross anatomy has not adequately incorporated modern embryological findings; consequently, the current understanding has often been incompatible with recent discoveries from molecular studies. Notably, the traditional epaxial and hypaxial muscle distinction, and their corresponding innervation by the dorsal and ventral rami of the spinal nerve, do not correspond to the primaxial and abaxial muscle distinction, defined by the mesodermal lineages of target tissues. To resolve the disagreement between adult anatomy and embryology, we here propose a novel hypothetical model of spinal nerve ramification. Our model is based on the previously unknown developmental process of the intercostal nerves. Observations of these nerves in the mouse embryos revealed that the intercostal nerves initially had superficial and deep ventral branches, which is contrary to the general perception of a single ventral branch. The initial dual innervation pattern later changes into an adult-like single branch pattern following the retraction of the superficial branch. The modified intercostal nerves consist of the canonical ventral branches and novel branches that run on the muscular surface of the thorax, which sprout from the lateral cutaneous branches. We formulated the embryonic branching pattern into the hypothetical ramification model of the human spinal nerve so that the branching pattern is compatible with the developmental context of the target muscles. In our model, every spinal nerve consists of three components: (1) segmental branches that innervate the primaxial muscles, including the dorsal rami, and short branches and long superficial anterior branches from the ventral rami; (2) plexus-forming intramural branches, the serial homolog of the canonical intercostal nerves, which innervate the abaxial portion of the body wall; and (3) plexus-forming extramural branches, the series of novel branches located outside of the body wall, which innervate the girdle and limb muscles. The selective elaboration or deletion of each component successfully explains the reasoning for the standard morphology and variability of the spinal nerve. Therefore, our model brings a novel understanding of spinal nerve development and valuable information for basic and clinical sciences regarding the diverse branching patterns of the spinal nerve.

Elbow valgus stability of the transverse bundle of the ulnar collateral ligament.

BACKGROUND: The purpose of this study was to clarify elbow valgus stability of the transverse bundle (TB). We hypothesized that the transverse bundle is involved in elbow valgus stability. METHODS: Twelve elbows of six Japanese Thiel-embalmed cadavers were evaluated. The skin, subcutaneous tissue and origin of forearm flexors were removed from about 5 cm proximal to the elbow to about 5 cm distal to the elbow, and the ulnar collateral ligament was dissected (intact state). The cut state was defined as the state when the TB was cut in the middle. The joint space of the humeroulnar joint (JS) was measured in the intact state and then in the cut state. With the elbow flexed to 30°, elbow valgus stress was gradually increased to 30, 60 N using the Telos Stress Device, and the JS was measured by ultrasonography under each load condition. Paired t-testing was performed to compare the JS between the intact and cut states under each load. RESULTS: No significant difference in JS was identified between the intact and cut state at start limb position. The JS was significantly higher in the cut state than in the intact state at both 30 N and 60 N. CONCLUSION: The findings from this study suggested that the TB may be involved in elbow valgus stability.

Conformal and Stoichiometric Chemical Vapor Deposition of Silicon Carbide onto Ultradeep Heterogeneous Micropores by Controlling the Initial Nucleation Stage.

Conformal chemical vapor deposition (CVD) of silicon carbide (SiC) from methyltrichlorosilane (MTS) and hydrogen (H2) onto high-aspect-ratio (HAR; typically >100:1) three-dimensional features has been a challenge in the fabrication of ceramic matrix composites. In this study, the impact of heterogeneous underlayers on the initial nucleation of SiC-CVD was studied using HAR (1000:1) microchannels with a tailored wetting underlayer of Si(100) and dewetting underlayers of thermally formed amorphous silicon dioxide (a-SiO2) and turbostratic boron nitride (t-BN). Incubation periods were distributed in the microchannels on a-SiO2 and t-BN underlayers, with the longest period of 70 min found at the feature-bottom due to a decreased concentration (C) of film-forming species. The longer incubation periods with more dewetting underlayers arose due to demoted initial nucleation. Prolonged incubation at the feature bottom led to poor conformality because thick films had already formed at the inlet when film formation began at the feature bottom. The incubation periods were eliminated by increasing the supply of MTS/H2, in accordance with classical heterogeneous nucleation theory. In the meantime, carbon-rich SiC films formed in the vicinity of dewetting a-SiO2 and t-BN underlayers at the feature bottoms, with greater carbon segregation on more dewetting underlayers. This was probably due to the deposition of pyrocarbons (CH4, C2H2, and/or C2H4) generated from MTS/H2 in the gas phase. Decreasing the temperature (T) from 1000 to 900 °C prevented carbon-rich film formation, and the expected deposition rate of pyrocarbon decreased to 0.6% for the case of CH4. A higher C of MTS/H2 combined with a lower T enabled conformal and stoichiometric film formation on the heterogeneous HAR features.

Contributions of the Third and Fourth Digits and the Second and Fifth Digits of the Flexor Digitorum Superficialis Muscle to Elbow Valgus Stability.

BACKGROUND: Thiel cadavers have been reported to have lifelike flexibility and mechanical properties, but whether they are useful for measurement of the ulnohumeral joint space (JS) is unclear. The contributions of the third and fourth digits and the second and fifth digits of the flexor digitorum superficialis (FDS) to elbow valgus stability are also unknown. PURPOSE: To (1) clarify whether Thiel cadavers can be used for JS measurement on ultrasound and (2) identify the contributions to valgus stability of the third and fourth digits and the second and fifth digits of the FDS. STUDY DESIGN: Descriptive laboratory study. METHODS: In experiment 1 (12 elbows from human volunteers and 12 elbows from Thiel cadavers), valgus stress was increased gradually from 0 to 30 to 60 N, and the JS was compared on ultrasound between groups at each load. In experiment 2 (13 elbows from Thiel cadavers), specimens were divided into 2 groups, and the JS was measured for group 1 with the FDS intact, with tendinous insertions of the third and fourth digits cut (3/4-cut state), and with tendinous insertions of all fingers cut (all-cut state); and for group 2 at intact FDS, with tendinous insertions of the second and fifth digits cut (2/5-cut state), and at all-cut. RESULTS: In experiment 1, the rate of change of the JS increased significantly with elbow valgus stress in both humans and Thiel cadavers, with no significant difference between groups. In experiment 2, the JS was significantly greater in the 3/4- and 2/5-cut states compared with the intact state at both 30 N (Δ3/4-cut vs intact = 0.23 mm [P = .01]; Δ2/5-cut vs intact = 0.32 mm [P = .02]) and 60 N (Δ3/4-cut vs intact = 0.33 mm [P = .002]; Δ2/5-cut vs intact = 0.37 mm [P = .04]). There was no significant difference in JS measurements between the 3/4- and 2/5-cut states at any load. CONCLUSION: Thiel cadavers showed JS changes similar to those of humans when valgus stress was applied. The third and fourth digits and the second and fifth digits of the FDS were involved in valgus stability, and there was no difference in their respective contributions. CLINICAL RELEVANCE: This study may help in identifying function of the FDS based on structure.

Development of fin-innervating motor neurons after peripheral target removal in medaka fish.

Peripheral targets regulate the development and survival of the nerve centers that serve them, because the elimination of the target normally results in massive death of the developing neurons that innervate it. This widely accepted theory appears to be well supported by developing limbs and their innervation in tetrapods, but it is unclear whether this concept applies to primitive vertebrates that have paired appendages. In this study, we examined the development of spinal motor neurons following pectoral fin bud removal (FBR) in medaka fish. After FBR, motor axons initially extended to the plexus region in a morphologically normal pattern. During the period of fin innervation, motor axons in the FBR-medaka failed to form the normal brachial plexus and elongated ventrally toward the abdominal region. In the ventral horn that would normally innervate the pectoral fin, however, neurons did not undergo cell death following FBR. There were no differences in the numbers of axons in the ventral roots between the FBR and control sides. Motor neuron markers, RALDH2 and FOXP1, that are expressed in limb-innervating motor neurons in the lateral motor column in tetrapods, were also expressed in the ventral horns of both the control and FBR sides in medaka fish. These results suggest that, although both tetrapod and medaka motor neurons share the same molecular characteristics for innervating paired appendages, the fates of neurons differ following the removal of their peripheral target. Therefore, the relationship between the peripheral target and its nerve center may be altered among vertebrates.

Porous Membranes as Sacrificial Layers Enabling Conformal Chemical Vapor Deposition Involving Multiple Film-Forming Species.

We propose a new, concise method for conformal chemical vapor deposition (CVD) using sacrificial layers (SLs) to fill three-dimensional features with microscopic pores. SLs are porous membranes (e.g., ceramic felts) that filter film-forming species having high sticking-probability (η). CVD processes with multiple film-forming species generally suffer from poor conformality due to preferential film deposition at the inlets of features by the high-η species, such as reactive intermediates. An SL traps such high-η species before they reach the target features and selectively supplies film-forming species with lower η (e.g., source precursors or stable intermediates) that enables conformal film deposition. Here the trapping efficiency of an SL was predicted and a procedure for designing an optimal SL was established. The procedure was demonstrated by CVD of silicon carbide (SiC) with multiple film-forming species of high-η species (η = 8.0 × 10-3) and lower-η species (η = 5.9 × 10-5 and 2.2 × 10-7). The trapping of 99.2% of incident high-η species was achieved with an optimized SL, wherein the deposition rate (m/s) contribution by high-η species declined from 0.546 at the SL inlet to 0.014 at its outlet. Finally, using these optimized SLs, SiC-CVD filling of micron-scale trenches was demonstrated with an aspect-ratio of 16:1.

Novel concept for the epaxial/hypaxial boundary based on neuronal development.

Trunk muscles in vertebrates are classified as either dorsal epaxial or ventral hypaxial muscles. Epaxial and hypaxial muscles are defined as muscles innervated by the dorsal and ventral rami of spinal nerves, respectively. Each cluster of spinal motor neurons passing through dorsal rami innervates epaxial muscles, whereas clusters traveling on the ventral rami innervate hypaxial muscles. Herein, we show that some motor neurons exhibiting molecular profiles for epaxial muscles follow a path in the ventral rami. Dorsal deep-shoulder muscles and some body wall muscles are defined as hypaxial due to innervation via the ventral rami, but a part of these ventral rami has the molecular profile of motor neurons that innervate epaxial muscles. Thus, the epaxial and hypaxial boundary cannot be determined simply by the ramification pattern of spinal nerves. We propose that, although muscle innervation occurs via the ventral rami, dorsal deep-shoulder muscles and some body wall muscles represent an intermediate group that lies between epaxial and hypaxial muscles.

The fornix acts as a permissive corridor for septal neuron migration beyond the diencephalic-telencephalic boundary.

Neuronal migration is essential for constructing functional neural networks. Two posterior septal (PS) nuclei, the triangular septal nucleus and bed nuclei of the anterior commissure, are involved in fear and anxiety. During development, glutamatergic PS neurons undergo long-distance rostrodorsal migration from the thalamic eminence (TE) of the diencephalon, then settle in the caudalmost telencephalon. However, the developmental behavior of PS neurons and the guidance structures facilitating their migration remain unknown. We previously demonstrated the migration of PS neurons along the fornix, a major efferent pathway from the hippocampal formation. Here, we show that the postcommissural fornix is essential for PS neuron migration which is largely confined to its axonal tract, which grows in the opposite direction as PS neuron migration. Fornical axons reach the TE prior to initiation of PS neuron rostrodorsal migration. Ectopic expression of Semaphorin 3 A in the dorsomedial cortex resulted in defective fornix formation. Furthermore, loss of the postcommissural fornix stalled PS neuron migration resulting in abnormal accumulation near their origin. This suggests that PS neurons utilize the postcommissural fornix as a permissive corridor during migration beyond the diencephalic-telencephalic boundary. This axonal support is essential for the functional organization of the heterogeneous septal nuclear complex.

Development of a mouse nerve-transfer model for brachial plexus injury.

Nerve transfer involves the use of a portion of a healthy nerve to repair an injured nerve, and the process has been used to alleviate traumatic brachial plexus injuries in humans. Study of the neural mechanisms that occur during nerve transfer, however, requires the establishment of reliable experimental models. In this study, we developed an ulnar-musculocutaneous nerve-transfer model wherein the biceps muscle of a mouse was re-innervated using a donor ulnar nerve. Similar muscle action potentials were detected in both the end-to-end suture of the transected nerve (correctrepair) group and the ulnar-musculocutaneous nerve-transfer group. Also, re-innervated acetylcholine receptor (AChR) clusters and muscle spindles were observed in both procedures. There were fewer re-innervated AChR clusters in the nerve transfer group than in the correct repair group at 4 weeks, but the numbers were equal at 24 weeks following surgery. Thus, our ulnar-musculocutaneous nerve-transfer model allowed physiological and morphological evaluation for re-innervation process in mice and revealed the delay of this process during nerve transfer procedure. This model will provide great opportunities to study regeneration, re-innervation, and functional recovery induced via nerve transfer procedures.

Diencephalic progenitors contribute to the posterior septum through rostral migration along the hippocampal axonal pathway.

Septal nuclei are telencephalic structures associated with a variety of brain functions as part of the limbic system. The two posterior septal nuclei, the triangular septal nucleus (TS) and the bed nuclei of the anterior commissure (BAC), are involved in fear and anxiety through their projections to the medial habenular nucleus. However, the development of both the TS and BAC remains unclear. Here, we found a novel caudal origin and putative migratory stream of mouse posterior septal neurons arising from the thalamic eminence (TE), a transient developmental structure at the rostral end of the rodent diencephalon. TE-derived cells, which have glutamatergic identity, migrated rostrally and entered the telencephalic territory by passing beneath the third ventricle. Subsequently, they turned dorsally toward the posterior septum. We also observed that TS and BAC neurons in the postnatal septum were labeled with GFP by in utero electroporation into the TE, suggesting a shared origin. Furthermore, TE-derived septal neurons migrated along the fornix, an efferent pathway from the hippocampus. These results demonstrate that posterior septal neurons have a distinct extratelencephalic origin from other septal nuclei. This heterogeneous origin may contribute to neuronal diversity of the septal nuclear complex.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern.

Basic Study on Visibility and Water Equivalency of a New Colorless Transparent Bolus for Electron Radiotherapy.

Boluses used in electron radiotherapy need to have radiation field visibility and water equivalence. In this report, we have examined field visibility and water equivalence of a new colorless transparent bolus. We examined field visibility, water equivalence, and dose profile. Field visibility was evaluated by comparison to conventional bolus. Water equivalence was investigated by a measured fluence scaling factor. The dose profile was measured by using radiochromic film with the bolus and an ionization chamber in water. We confirmed that the irradiation field could clearly be seen through the transparent colorless bolus. The bolus did not cast a field edge as compared with the conventional bolus. The fluence scaling factor was less than 0.8% as compared to water. We confirmed that the colorless transparent bolus was treated as a water equivalent material. The percentage depth dose (PDD) measured by using radiochromic film with the bolus matched the PDD measured with an ionization chamber in water. R50 was less than 1 mm as compared to PDD measured with an ionization chamber. It was confirmed that the colorless transparent bolus can use to set up patient without losing visibility on flat ground planes. The fluence scaling factor and dose profile measured by using the bolus matched the results measured in water. Therefore, the new colorless transparent bolus has feasibility to improve patient setup efficiency and can improve calculation accuracy by using the fluence scaling factor.

Endoplasmic Reticulum-Localized Transmembrane Protein Dpy19L1 Is Required for Neurite Outgrowth.

The endoplasmic reticulum (ER), including the nuclear envelope, is a continuous and intricate membrane-bound organelle responsible for various cellular functions. In neurons, the ER network is found in cell bodies, axons, and dendrites. Recent studies indicate the involvement of the ER network in neuronal development, such as neuronal migration and axonal outgrowth. However, the regulation of neural development by ER-localized proteins is not fully understood. We previously reported that the multi-transmembrane protein Dpy19L1 is required for neuronal migration in the developing mouse cerebral cortex. A Dpy19L family member, Dpy19L2, which is a causative gene for human Globozoospermia, is suggested to act as an anchor of the acrosome to the nuclear envelope. In this study, we found that the patterns of exogenous Dpy19L1 were partially coincident with the ER, including the nuclear envelope in COS-7 cells at the level of the light microscope. The reticular distribution of Dpy19L1 was disrupted by microtubule depolymerization that induces retraction of the ER. Furthermore, Dpy19L1 showed a similar distribution pattern with a ER marker protein in embryonic mouse cortical neurons. Finally, we showed that Dpy19L1 knockdown mediated by siRNA resulted in decreased neurite outgrowth in cultured neurons. These results indicate that transmembrane protein Dpy19L1 is localized to the ER membrane and regulates neurite extension during development.

Developmental origin of the clavicle, and its implications for the evolution of the neck and the paired appendages in vertebrates.

In fish, the pectoral appendage is adjacent to the head, but during vertebrate evolution a long neck region emerged via caudal relocation of the pectoral appendage. The pectoral appendage is comprised of endochondral portions, such as the humerus and the scapula, and a dermal portion, such as the clavicle, that contributes to the shoulder girdle. In the search for clues to the mechanism of the caudal relocation of the pectoral appendage, the cell lineage of the rostral lateral plate mesoderm was analyzed in chickens. It was found that, despite the long neck region in chickens, the origin of the clavicle attached to the head mesoderm ranged between 1 and 14 somite levels. Because the pectoral limb bud and the endochondral pectoral appendage developed on 15-20 and 15-24 somite levels, respectively, the clavicle-forming region corresponds to the embryonic neck, which suggests that the relocation would have been executed by the expansion of the source of the clavicle. The rostral portion of the clavicle-forming region overlaps the source of the cucullaris muscle, embraces the pharyngeal arches caudally, and can be experimentally replaced with the head mesoderm to form the cucullaris muscle, which implies that the mesodermal portion could have been the head mesoderm and that the clavicle would have developed at the head/trunk boundary. The link between the head mesoderm and the presumptive clavicle appears to have been the developmental constraint needed to create the evolutionarily conserved musculoskeletal connectivities characterizing the gnathostome neck. In this sense, the dermal girdle of the ganathostomes would represent the wall of the branchial chamber into which the endochondral pectoral appendage appears to have attached since its appearance in evolution.

Evidence from cyclostomes for complex regionalization of the ancestral vertebrate brain.

The vertebrate brain is highly complex, but its evolutionary origin remains elusive. Because of the absence of certain developmental domains generally marked by the expression of regulatory genes, the embryonic brain of the lamprey, a jawless vertebrate, had been regarded as representing a less complex, ancestral state of the vertebrate brain. Specifically, the absence of a Hedgehog- and Nkx2.1-positive domain in the lamprey subpallium was thought to be similar to mouse mutants in which the suppression of Nkx2-1 leads to a loss of the medial ganglionic eminence. Here we show that the brain of the inshore hagfish (Eptatretus burgeri), another cyclostome group, develops domains equivalent to the medial ganglionic eminence and rhombic lip, resembling the gnathostome brain. Moreover, further investigation of lamprey larvae revealed that these domains are also present, ruling out the possibility of convergent evolution between hagfish and gnathostomes. Thus, brain regionalization as seen in crown gnathostomes is not an evolutionary innovation of this group, but dates back to the latest vertebrate ancestor before the divergence of cyclostomes and gnathostomes more than 500 million years ago.