Hox2 Genes Are Required for Tonotopic Map Precision and Sound Discrimination in the Mouse Auditory Brainstem.

Tonotopy is a hallmark of auditory pathways and provides the basis for sound discrimination. Little is known about the involvement of transcription factors in brainstem cochlear neurons orchestrating the tonotopic precision of pre-synaptic input. We found that in the absence of Hoxa2 and Hoxb2 function in Atoh1-derived glutamatergic bushy cells of the anterior ventral cochlear nucleus, broad input topography and sound transmission were largely preserved. However, fine-scale synaptic refinement and sharpening of isofrequency bands of cochlear neuron activation upon pure tone stimulation were impaired in Hox2 mutants, resulting in defective sound-frequency discrimination in behavioral tests. These results establish a role for Hox factors in tonotopic refinement of connectivity and in ensuring the precision of sound transmission in the mammalian auditory circuit.

Assembly of the auditory circuitry by a Hox genetic network in the mouse brainstem.

Rhombomeres (r) contribute to brainstem auditory nuclei during development. Hox genes are determinants of rhombomere-derived fate and neuronal connectivity. Little is known about the contribution of individual rhombomeres and their associated Hox codes to auditory sensorimotor circuitry. Here, we show that r4 contributes to functionally linked sensory and motor components, including the ventral nucleus of lateral lemniscus, posterior ventral cochlear nuclei (VCN), and motor olivocochlear neurons. Assembly of the r4-derived auditory components is involved in sound perception and depends on regulatory interactions between Hoxb1 and Hoxb2. Indeed, in Hoxb1 and Hoxb2 mutant mice the transmission of low-level auditory stimuli is lost, resulting in hearing impairments. On the other hand, Hoxa2 regulates the Rig1 axon guidance receptor and controls contralateral projections from the anterior VCN to the medial nucleus of the trapezoid body, a circuit involved in sound localization. Thus, individual rhombomeres and their associated Hox codes control the assembly of distinct functionally segregated sub-circuits in the developing auditory brainstem.

Body plan of turtles: an anatomical, developmental and evolutionary perspective.

The evolution of the turtle shell has long been one of the central debates in comparative anatomy. The turtle shell consists of dorsal and ventral parts: the carapace and plastron, respectively. The basic structure of the carapace comprises vertebrae and ribs. The pectoral girdle of turtles sits inside the carapace or the rib cage, in striking contrast to the body plan of other tetrapods. Due to this topological change in the arrangement of skeletal elements, the carapace has been regarded as an example of evolutionary novelty that violates the ancestral body plan of tetrapods. Comparing the spatial relationships of anatomical structures in the embryos of turtles and other amniotes, we have shown that the topology of the musculoskeletal system is largely conserved even in turtles. The positional changes seen in the ribs and pectoral girdle can be ascribed to turtle-specific folding of the lateral body wall in the late developmental stages. Whereas the ribs of other amniotes grow from the axial domain to the lateral body wall, turtle ribs remain arrested axially. Marginal growth of the axial domain in turtle embryos brings the morphologically short ribs in to cover the scapula dorsocaudally. This concentric growth appears to be induced by the margin of the carapace, which involves an ancestral gene expression cascade in a new location. These comparative developmental data allow us to hypothesize the gradual evolution of turtles, which is consistent with the recent finding of a transitional fossil animal, Odontochelys, which did not have the carapace but already possessed the plastron.

Hepatocyte growth factor is crucial for development of the carapace in turtles.

Turtles are characterized by their shell, composed of a dorsal carapace and a ventral plastron. The carapace first appears as the turtle-specific carapacial ridge (CR) on the lateral aspect of the embryonic flank. Accompanying the acquisition of the shell, unlike in other amniotes, hypaxial muscles in turtle embryos appear as thin threads of fibrous tissue. To understand carapacial evolution from the perspective of muscle development, we compared the development of the muscle plate, the anlage of hypaxial muscles, between the Chinese soft-shelled turtle, Pelodiscus sinensis, and chicken embryos. We found that the ventrolateral lip (VLL) of the thoracic dermomyotome of P. sinensis delaminates early and produces sparse muscle plate in the lateral body wall. Expression patterns of the regulatory genes for myotome differentiation, such as Myf5, myogenin, Pax3, and Pax7 have been conserved among amniotes, including turtles. However, in P. sinensis embryos, the gene hepatocyte growth factor (HGF), encoding a regulatory factor for delamination of the dermomyotomal VLL, was uniquely expressed in sclerotome and the lateral body wall at the interlimb level. Implantation of COS-7 cells expressing a HGF antagonist into the turtle embryo inhibited CR formation. We conclude that the de novo expression of HGF in the turtle mesoderm would have played an innovative role resulting in the acquisition of the turtle-specific body plan.

Lineage-specific duplication and loss of pepsinogen genes in hominoid evolution.

Fourteen different pepsinogen-A cDNAs and one pepsinogen-C cDNA have been cloned from gastric mucosa of the orangutan, Pongo pygmaeus. Encoded pepsinogens A were classified into two groups, i.e., types A1 and A2, which are different in acidic character. The occurrence of 9 and 5 alleles of A1 and A2 genes (at least 5 and 3 loci), respectively was anticipated. Respective orthologous genes are present in the chimpanzee genome although their copy numbers are much smaller than those of the orangutan genes. Only A1 genes are present in the human probably due to the loss of the A2 gene. Molecular phylogenetic analyses showed that A1 and A2 genes diverged before the speciation of great hominoids. Further reduplications of respective genes occurred several times in the orangutan lineage, with much higher frequencies than those occurred in the chimpanzee and human lineages. The rates of non-synonymous substitutions were higher than those of synonymous ones in the lineage of A2 genes, implying the contribution of the positive selection on the encoded enzymes. Several sites of pepsin moieties were indeed found to be under positive selection, and most of them locate on the surface of the molecule, being involved in the conformational flexibility. Deduced from the known genomic structures of pepsinogen-A genes of primates and other mammals, the duplication/loss were frequent during their evolution. The extreme multiplication in the orangutan might be advantageous for digestion of herbaceous foods due to the increase in the level of enzymes in stomach and the diversification of enzyme specificity.

Differences in the P1' substrate specificities of pepsin A and chymosin.

Porcine pepsin A and bovine chymosin are typical models of aspartic proteinases. The hydrolytic specificities of these proteinases, along with those of human pepsin A and monkey chymosin, were investigated with 29 peptide substrates that included various P1' variants of seven parent peptides. From these peptides, AFPLEF downward arrow FREL was preferred by pepsin A and chymosin, while its P1' variant, AFPLEF downward arrow EREL was preferred by bovine chymosin. Porcine and human pepsin A showed similar hydrolytic specificities, strongly preferring a hydrophobic/aromatic residue at P1' of any type of peptide. This specificity is well explained by the very hydrophobic nature of the S1' subsite that consists of Tyr(189), Ile(213), Ile(300), Met(289), Val/Leu(291) and Leu(298). The first three residues are well conserved in pepsin family enzymes. Although bovine and monkey chymosin showed similar P1' specificity, bovine chymosin preferred peptides having Glu at P1', while monkey chymosin preferred peptides having Lys at P1'. The dual characteristics of chymosin are due to the occurrence of polar/charged residues in the S1' subsite, such as Glu/Asp(289), Gln(298) and Lys/Gln(299), which are different from the S1' subsite of pepsin A. Molecular models suggest that Glu in position 289 of bovine chymosin and Asp in position 289 of monkey chymosin are responsible for the difference in P1' specificities between the chymosins.

Hox genes in neural patterning and circuit formation in the mouse hindbrain.

The mammalian hindbrain is the seat of regulation of several vital functions that involve many of the organ systems of the body. Such functions are controlled through the activity of intricate arrays of neuronal circuits and connections. The establishment of ordered patterns of neuronal specification, migration, and axonal topographic connectivity during development is crucial to build such a complex network of circuits and functional connectivity in the mature hindbrain. The early development of the vertebrate hindbrain proceeds according to a fundamental metameric partitioning along the anteroposterior axis into cellular compartments known as rhombomeres. Such an organization has been highly conserved in vertebrate evolution and has a fundamental impact on the hindbrain adult structure, nuclear organization, and connectivity. Here, we review the cellular and molecular mechanisms underlying hindbrain neuronal circuitry in the mouse, with a specific focus on the role of the homeodomain transcription factors of the Hox gene family. The Hox genes are crucial determinants of rhombomere segmental identity and anteroposterior patterning. However, recent findings suggest that, in addition to their well-known roles at early embryonic stages, the Hox genes may play important roles also in later aspect of neuronal circuit development, including stereotypic neuronal migration, axon pathfinding, and topographic mapping of connectivity.

Evolution of the turtle body plan by the folding and creation of new muscle connections.

The turtle shell offers a fascinating case study of vertebrate evolution, based on the modification of a common body plan. The carapace is formed from ribs, which encapsulate the scapula; this stands in contrast to the typical amniote body plan and serves as a key to understanding turtle evolution. Comparative analyses of musculoskeletal development between the Chinese soft-shelled turtle and other amniotes revealed that initial turtle development conforms to the amniote pattern; however, during embryogenesis, lateral rib growth results in a shift of elements. In addition, some limb muscles establish new turtle-specific attachments associated with carapace formation. We propose that the evolutionary origin of the turtle body plan results from heterotopy based on folding and novel connectivities.

Optogenetic Dissection of Neuronal Circuits in Zebrafish using Viral Gene Transfer and the Tet System.

The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

Hox gene expression patterns in Lethenteron japonicum embryos--insights into the evolution of the vertebrate Hox code.

The Hox code of jawed vertebrates is characterized by the colinear and rostrocaudally nested expression of Hox genes in pharyngeal arches, hindbrain, somites, and limb/fin buds. To gain insights into the evolutionary path leading to the gnathostome Hox code, we have systematically analyzed the expression pattern of the Hox gene complement in an agnathan species, Lethenteron japonicum (Lj). We have isolated 15 LjHox genes and assigned them to paralogue groups (PG) 1-11, based on their deduced amino acid sequences. LjHox expression during development displayed gnathostome-like spatial patterns with respect to the PG numbers. Specifically, lamprey PG1-3 showed homologous expression patterns in the rostral hindbrain and pharyngeal arches to their gnathostome counterparts. Moreover, PG9-11 genes were expressed specifically in the tailbud, implying its posteriorizing activity as those in gnathostomes. We conclude that these gnathostome-like colinear spatial patterns of LjHox gene expression can be regarded as one of the features already established in the common ancestor of living vertebrates. In contrast, we did not find evidence for temporal colinearity in the onset of LjHox expression. The genomic and developmental characteristics of Hox genes from different chordate species are also compared, focusing on evolution of the complex body plan of vertebrates.

On the carapacial ridge in turtle embryos: its developmental origin, function and the chelonian body plan.

The chelonian carapace is composed of dorsolaterally expanded ribs; an evolutionary change in the rib-patterning program is assumed to be related to this novelty. Turtle embryos exhibit a longitudinal ridge called the carapacial ridge (CR) on the flank, and its histological resemblance to the apical ectodermal ridge of the limb bud implies its inductive activity in the unique patterning of the ribs. We studied the Chinese soft-shelled turtle, Pelodiscus sinensis, and confirmed by labeling with a lipophilic dye, DiI, that the CR contains the somite-derived dermis and that it is a unique structure among amniotes. Using electroporation of a dominant-negative form of LEF-1, the CR-specific gene, we showed that CR-specific genes function in the growth and maintenance of the CR. Microcauterization or implantation of the CR did not change the dorsoventral pattern of the ribs, and only their fan-shaped pattern was arrested by CR removal. We conclude that the CR is a true embryonic novelty among amniotes and, because of the specific expression of regulatory genes, it functions in the marginal growth of the carapacial primordium, thereby inducing the fan-shaped arrangement of the ribs.

Rodent monophyly deduced from the unique gastric proteinase constitution and molecular phylogenetic analyses using pepsinogen-C cDNA sequences.

Pepsinogens are zymogens of pepsins, the gastric digestive proteinases. Although pepsinogen A is predominant in most mammalian species hitherto known, pepsinogen C is expressed exclusively and the lack of pepsinogen A is evidenced in the rat and guinea pig. Furthermore, in these two rodents, considerable amount of procathepsin E is also expressed in gastric mucosa although it is almost undetectable in other mammals. In this paper, in order to clarify whether such unique gastric proteinase constitution is common among rodents, we carried out purification and characterization of gastric proteinases, and molecular cloning of pepsinogen-C cDNAs from several rodent species including the degu and coypu. Pepsinogen C and procathepsin E were isolated but pepsinogen A was undetectable in the rodents, leading to the conclusion that that rodents commonly share the unique gastric proteinase constitution. This feature could be treated as a new "molecular synapomorphy", supporting strongly monophyly of the order Rodentia. From the molecular phylogenetic analyses of pepsinogen-C cDNA sequences, monophyly of the order Rodentia was also supported by the analyses with high statistic reliabilities.

Evolution of the vertebral formulae in mammals: a perspective on developmental constraints.

Developmental constraints refer to biases that limit phenotypic changes during evolution. To examine the contribution of developmental constraints in the evolution of vertebrate morphology, we analyzed the distribution pattern of mammalian vertebral formulae. Data on mammalian vertebral formulae were collected from the Descriptive Catalogue of the Osteological Series Contained in the Museum of the Royal College of Surgeons of England by Richard Owen (1853) and were plotted onto the most reliable mammalian phylogenetic tree based on recent molecular studies. In addition to the number of cervical vertebrae that is almost fixed to 7, we found that the number of thoracolumbar vertebrae tends to be 19 in many groups of mammals. Since fidelity of the number of thoracolumbar vertebrae was also completely maintained in Monotremata and Marsupialia, we presumed that thoracolumbar vertebral number as well as cervical vertebral number might have been fixed in the primitive mammalian lineage. On the basis of primitive vertebral formulae, we could clarify the polarity of evolution and identify several deviations from the primitive states during the mammalian evolution. The changes in the vertebral formulae in eutherian mammals seem to be lineage-specific, such that most species in Carnivora have 20 instead of 19 thoracolumbar vertebrae. Because such lineage-specific vertebral formulae contrast with the estimated distribution pattern on the assumption of evolution only through the selective pressure, we concluded that developmental constraints played an important role in the evolution of mammalian vertebral formulae.

Primary structure, unique enzymatic properties, and molecular evolution of pepsinogen B and pepsin B.

Purification of pepsinogen B from dog stomach was achieved. Activation of pepsinogen B to pepsin B is likely to proceed through a one-step pathway although the rate is very slow. Pepsin B hydrolyzes various peptides including beta-endorphin, insulin B chain, dynorphin A, and neurokinin A, with high specificity for the cleavage of the Phe-X bonds. The stability of pepsin B in alkaline pH is noteworthy, presumably due to its less acidic character. The complete primary structure of pepsinogen B was clarified for the first time through the molecular cloning of the respective cDNA. Molecular evolutional analyses show that pepsinogen B is not included in other known pepsinogen groups and constitutes an independent cluster in the consensus tree. Pepsinogen B might be a sister group of pepsinogen C and the divergence of these two zymogens seems to be the latest event of pepsinogen evolution.