Segmentation of the vertebrate body axis is established in the embryo by formation of somites, which give rise to the axial muscles (myotome) and vertebrae (sclerotome). To allow a muscle to attach to two successive vertebrae, the myotome and sclerotome must be repositioned by half a segment with respect to each other. Two main models have been put forward: 'resegmentation' proposes that each half-sclerotome joins with the half-sclerotome from the next adjacent somite to form a vertebra containing cells from two successive somites on each side of the midline. The second model postulates that a single vertebra is made from a single somite and that the sclerotome shifts with respect to the myotome. There is conflicting evidence for these models, and the possibility that the mechanism may vary along the vertebral column has not been considered. Here we use DiI and DiO to trace somite contributions to the vertebrae in different axial regions in the chick embryo. We demonstrate that vertebral bodies and neural arches form by resegmentation but that sclerotome cells shift in a region-specific manner according to their dorsoventral position within a segment. We propose a 'resegmentation-shift' model as the mechanism for amniote vertebral patterning.

Embryonic patterning of the mouse during gastrulation and early organogenesis engenders the specification of anterior versus posterior structures and body laterality by the interaction of signalling and modulating activities. A group of cells in the mouse gastrula, characterised by the expression of a repertoire of "organiser" genes, acts as a source and the conduit for allocation of the axial mesoderm, floor plate and definitive endoderm. The organiser and its derivatives provide the antagonistic activity that modulates WNT and TGFbeta signalling. Recent findings show that the organiser activity is augmented by morphogenetic activity of the extraembryonic and embryonic endoderm, suggesting embryonic patterning is not solely the function of the organiser.

The kidney is a model developmental system for understanding mesodermal patterning and organogenesis, a process that requires regional specification along multiple body axes, the proliferation and differentiation of progenitor cells, and integration with other tissues. Recent progress in the field has highlighted the essential roles of intrinsic nuclear factors and secreted signaling molecules in specifying renal epithelial stem cells and their self-renewal, in driving the complex dynamics of epithelial cell branching morphogenesis, and in nephron patterning. How these developments influence and advance our understanding of kidney development is discussed.

Cheyne-Stokes respiration (CSR) is a breathing pattern characterized by waxing and waning of breath volume and frequency, and is often recognized following stroke, when causal pathways are often obscure. We used an animal model to address the hypothesis that cerebral infarction is a mechanism for producing breathing instability. Fourteen male A/J mice underwent either stroke (n=7) or sham (n=7) procedure. Ventilation was measured using whole body plethysmography. Respiratory rate (RR), tidal volume (V(T)) and minute ventilation (V(e)) mean values and coefficient of variation were computed for ventilation and oscillatory behaviors. In addition, the ventilatory data were computationally fit to models to quantify autocorrelation, mutual information, sample entropy and a nonlinear complexity index. At the same time post-procedure, stroke when compared to sham animal breathing consisted of a lower RR and autocorrelation, higher coefficient of variation for V(T) and higher coefficient of variation for V(e). Mutual information and the nonlinear complexity index were higher in breathing following stroke which also demonstrated a waxing/waning pattern. The absence of stroke in the sham animals was verified anatomically. We conclude that ventilatory pattern following cerebral infarction demonstrated increased variability with increased nonlinear patterning and a waxing/waning pattern, consistent with CSR.

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (de(H)), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (a(t)) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the de(H) mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in de(H) that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in de(H)/de(H) animals. Construction of a targeted allele of Tbx15 confirmed that the de(H) phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

Pesticide exposure to the non target groups especially during embryonic development has quite often resulted in congenital malformations. A commercially available combination insecticide (Ci, 50% chlorpyrifos and 5% cypermethrin) is known to induce ventral body wall defects (VBWDs) wherein abdominal viscera protrude out of the ventral body wall. Herein, an attempt was made to understand the mechanistic insight into Ci induced VBWDs. For this, before incubation, the chick embryos were dosed with the test chemical and then at different developmental stages of incubation, they were monitored for the changes in the expression of certain genes, which are indispensable for the ventral body wall closure since they regulate the cell fate, proliferation and survival. Concurrently, histopathological changes during the embryonic development were examined to corroborate the above observations. The results of mRNA profiling revealed a significant downregulation of Shh on day 4 and upregulation on day 10, while bmp4, Pitx2, E-cadherin, Wnt11, Wnt6, Pxn, MyoD1, Caspase-3, AHR, Cyp3A4, showed a significant upregulation on day 4 and/or on day 10. N-cadherin, fgf8, bmp1 showed no significant changes. The possible means by which these skewed expression patterns of regulatory molecules culminated into the VBWD are discussed.

The patterning of an internal organ, like the heart, is little understood. Central to this patterning is the formation, or the acquisition, of an anteroposterior (A-P) axis. We have approached the question of how the heart tube acquires polarity in the zebrafish, Brachydanio rerio, which offers numerous advantages for studying cardiac morphogenesis. During the early stages of organogenesis in the fish, the heart tube lies in an A-P orientation with the venous end lying anteriorly and the arterial end lying posteriorly. High doses (10(-6)-10(-5)M) of retinoic acid (RA) cause truncation of the body axis, as they do in Xenopus. Low doses of retinoic acid (10(-8)-10(-7) M), which do not appear to affect the rest of the embryo, have pronounced effects upon heart tube morphogenesis, causing it to shrink progressively along the A-P axis. To investigate this further, we identified monoclonal antibodies that distinguish between the zebrafish cardiac chambers and used them to show that the RA-induced cardiac truncation always begins at the arterial end of the heart tube. There is a continuous gradient of sensitivity from the arterial to the venous end, such that increasing RA exposure causes the progressive and sequential deletion first of the bulbus arteriosus and then, in order, of the ventricle, the atrium, and the sinus venosus. As exposure increases, parts of chambers are deleted before entire chambers; thus, the sensitivity to RA appears to be independent of chamber boundaries. The analysis of the heart tube's sensitivity to RA and its timing suggest that polarity is established during or shortly after initial commitment to the cardiac lineage.

Vertebrate Hox clusters contain protein-coding genes that regulate body axis development and microRNA (miRNA) genes whose functions are not yet well understood. We overexpressed the Hox cluster microRNA miR-196 in zebrafish embryos and found four specific, viable phenotypes: failure of pectoral fin bud initiation, deletion of the 6th pharyngeal arch, homeotic aberration and loss of rostral vertebrae, and reduced number of ribs and somites. Reciprocally, miR-196 knockdown evoked an extra pharyngeal arch, extra ribs, and extra somites, confirming endogenous roles of miR-196. miR-196 injection altered expression of hox genes and the signaling of retinoic acid through the retinoic acid receptor gene rarab. Knocking down rarab mimicked the pectoral fin phenotype of miR-196 overexpression, and reporter constructs tested in tissue culture and in embryos showed that the rarab 3'UTR is a miR-196 target for pectoral fin bud initiation. These results show that a Hox cluster microRNA modulates development of axial patterning similar to nearby protein-coding Hox genes, and acts on appendicular patterning at least in part by modulating retinoic acid signaling.

Understanding the molecular interactions that lead to the establishment of the major body axes during embryogenesis is one of the main goals of developmental biology. Although the past two decades have revolutionized our knowledge about the genetic basis of these patterning processes, the list of genes involved in axis formation is unlikely to be complete. In order to identify new genes involved in the establishment of the dorsoventral (DV) axis during early stages of zebrafish embryonic development, we employed next generation sequencing for full transcriptome analysis of normal embryos and embryos lacking overt DV pattern. A combination of different statistical approaches yielded 41 differentially expressed candidate genes and we confirmed by in situ hybridization the early dorsal expression of 32 genes that are transcribed shortly after the onset of zygotic transcription. Although promoter analysis of the validated genes suggests no general enrichment for the binding sites of early acting transcription factors, most of these genes carry "bivalent" epigenetic histone modifications at the time when zygotic transcription is initiated, suggesting a "poised" transcriptional status. Our results reveal some new candidates of the dorsal gene regulatory network and suggest that a plurality of the earliest upregulated genes on the dorsal side have a role in the modulation of the canonical Wnt pathway.

Here we present a detailed statistical analysis of the discharge characteristics of mitral cells of the main olfactory bulb of urethane-anesthetized rats. Neurons were recorded from the mitral cell layer, and antidromically identified by stimuli applied to the lateral olfactory tract. All mitral cells displayed repeated, prolonged bursts of action potentials typically lasting >100 sec and separated by similarly long intervals; about half were completely silent between bursts. No such bursting was observed in nonmitral cells recorded in close proximity to mitral cells. Bursts were asynchronous among even adjacent mitral cells. The intraburst activity of most mitral cells showed strong entrainment to the spontaneous respiratory rhythm; similar entrainment was seen in some, but not all nonmitral cells. All mitral cells displayed a peak of excitability at ~25 msec after spikes, as reflected by a peak in the interspike interval distribution and in the corresponding hazard function. About half also showed a peak at about 6 msec, reflecting the common occurrence of doublet spikes. Nonmitral cells showed no such doublet spikes. Bursts typically increased in intensity over the first 20-30 sec of a burst, during which time doublets were rare or absent. After 20-30 sec (in cells that exhibited doublets), doublets occurred frequently for as long as the burst persisted, in trains of up to 10 doublets. The last doublet was followed by an extended relative refractory period the duration of which was independent of train length. In cells that were excited by application of a particular odor, responsiveness was apparently greater during silent periods between bursts than during bursts. Conversely in cells that were inhibited by a particular odor, responsiveness was only apparent when cells were active. Extensive raw (event timing) data from the cells, together with details of those analyses, are provided as supplementary material, freely available for secondary use by others.

Mechanical input shapes cell fate decisions during development and regeneration in many systems, yet the mechanisms of this cross-talk are often unclear. In regenerating Hydra tissue spheroids, periodic osmotically driven inflation and deflation cycles generate mechanical stimuli in the form of tissue stretching. Here, we demonstrate that tissue stretching during inflation is important for the appearance of the head organizer—a group of cells that secrete the Wnt3 ligand. Exploiting time series RNA expression profiles, we identify the up-regulation of Wnt signaling as a key readout of the mechanical input. In this system, the levels of Wnt3 expression correspond to the levels of stretching, and Wnt3 overexpression alone enables successful regeneration in the absence of mechanical stimulation. Our findings enable the incorporation of mechanical signals in the framework of Hydra patterning and highlight the broad significance of mechanochemical feedback loops for patterning epithelial lumens.

Individuals can vary substantially in size, but the proportions of their body plans are often maintained. We generated smaller zebrafish by removing 30% of their cells at the blastula stages and found that these embryos developed into normally patterned individuals. Strikingly, the proportions of all germ layers adjusted to the new embryo size within 2 hours after cell removal. As Nodal-Lefty signalling controls germ-layer patterning, we performed a computational screen for scale-invariant models of this activator-inhibitor system. This analysis predicted that the concentration of the highly diffusive inhibitor Lefty increases in smaller embryos, leading to a decreased Nodal activity range and contracted germ-layer dimensions. In vivo studies confirmed that Lefty concentration increased in smaller embryos, and embryos with reduced Lefty levels or with diffusion-hindered Lefty failed to scale their tissue proportions. These results reveal that size-dependent inhibition of Nodal signalling allows scale-invariant patterning.

While sleeping, many vertebrate groups alternate between at least two sleep stages: rapid eye movement and slow wave sleep1-4, in part characterized by wake-like and synchronous brain activity, respectively. Here we delineate neural and behavioural correlates of two stages of sleep in octopuses, marine invertebrates that evolutionarily diverged from vertebrates roughly 550 million years ago (ref. 5) and have independently evolved large brains and behavioural sophistication. 'Quiet' sleep in octopuses is rhythmically interrupted by approximately 60-s bouts of pronounced body movements and rapid changes in skin patterning and texture6. We show that these bouts are homeostatically regulated, rapidly reversible and come with increased arousal threshold, representing a distinct 'active' sleep stage. Computational analysis of active sleep skin patterning reveals diverse dynamics through a set of patterns conserved across octopuses and strongly resembling those seen while awake. High-density electrophysiological recordings from the central brain reveal that the local field potential (LFP) activity during active sleep resembles that of waking. LFP activity differs across brain regions, with the strongest activity during active sleep seen in the superior frontal and vertical lobes, anatomically connected regions associated with learning and memory function7-10. During quiet sleep, these regions are relatively silent but generate LFP oscillations resembling mammalian sleep spindles11,12 in frequency and duration. The range of similarities with vertebrates indicates that aspects of two-stage sleep in octopuses may represent convergent features of complex cognition.

When early canonical Wnt is experimentally inhibited, sea urchin embryos embody the concept of a Default Model in vivo because most of the ectodermal cell fates are specified as anterior neuroectoderm. Using this model, we describe here how the combination of orthogonally functioning anteroposterior Wnt and dorsoventral Nodal signals and their targeting transcription factors, FoxQ2 and Homeobrain, regulates the precise patterning of normal neuroectoderm, of which serotonergic neurons are differentiated only at the dorsal/lateral edge. Loss-of-function experiments revealed that ventral Nodal is required for suppressing the serotonergic neural fate in the ventral side of the neuroectoderm through the maintenance of foxQ2 and the repression of homeobrain expression. In addition, non-canonical Wnt suppressed homeobrain in the anterior end of the neuroectoderm, where serotonergic neurons are not differentiated. Canonical Wnt, however, suppresses foxQ2 to promote neural differentiation. Therefore, the three-dimensionally complex patterning of the neuroectoderm is created by cooperative signals, which are essential for the formation of primary and secondary body axes during embryogenesis.

Brain wiring depends on cells making highly localized and selective connections through surface protein-protein interactions, including those between NetrinGs and NetrinG ligands (NGLs). The NetrinGs are members of the structurally uncharacterized netrin family. We present a comprehensive crystallographic analysis comprising NetrinG1-NGL1 and NetrinG2-NGL2 complexes, unliganded NetrinG2 and NGL3. Cognate NetrinG-NGL interactions depend on three specificity-conferring NetrinG loops, clasped tightly by matching NGL surfaces. We engineered these NGL surfaces to implant custom-made affinities for NetrinG1 and NetrinG2. In a cellular patterning assay, we demonstrate that NetrinG-binding selectivity can direct the sorting of a mixed population of NGLs into discrete cell surface subdomains. These results provide a molecular model for selectivity-based patterning in a neuronal recognition system, dysregulation of which is associated with severe neuropsychological disorders.

The larval nervous system of the solitary tunicate Ciona is a simple model for the study of chordate neurodevelopment. The development and connectivity of the Ciona motor ganglion have been studied in fine detail, but how this important structure develops in other tunicates is not well known.

To assess evolutional changes in the expression pattern of Otx paralogues, expression analyses were undertaken in fugu, bichir, skate and lamprey. Together with those in model vertebrates, the comparison suggested that a gnathostome ancestor would have utilized all of Otx1, Otx2 and Otx5 paralogues in organizer and anterior mesendoderm for head development. In this animal, Otx1 and Otx2 would have also functioned in specification of the anterior neuroectoderm at presomite stage and subsequent development of forebrain/midbrain at somite stage, while Otx5 expression would have already been specialized in epiphysis and eyes. Otx1 and Otx2 functions in anterior neuroectoderm and brain of the gnathostome ancestor would have been differentially maintained by Otx1 in a basal actinopterygian and by Otx2 in a basal sarcopterygian. Otx5 expression in head organizer and anterior mesendoderm seems to have been lost in the teleost lineage after divergence of bichir, and also from the amniotes after divergence of amphibians as independent events. Otx1 expression was lost from the organizer in the tetrapod lineage. In contrast, in a teleost ancestor prior to whole genome duplication, Otx1 and Otx2 would have both been expressed in the dorsal margin of blastoderm, embryonic shield, anterior mesendoderm, anterior neuroectoderm and forebrain/midbrain, at respective stages of head development. Subsequent whole genome duplication and the following genome changes would have caused different Otx paralogue usages in each teleost lineage. Lampreys also have three Otx paralogues; their sequences are highly diverged from gnathostome cognates, but their expression pattern is well related to those of skate Otx cognates.

Spatial polarity cues in animals are used repeatedly during development for many processes, including cell fate determination, cell migration, and axon guidance. In Caenorhabditis elegans, the body wall muscle extends the length of the animal in four distinct quadrants and generates an UNC-129/TGF-β-related signal that is much higher in the dorsal two muscle quadrants compared to their ventral counterparts. This pattern of unc-129 expression requires the activity of the proposed transcriptional repressor UNC-130/FOXD whose body wall muscle activity is restricted to the ventral two body wall muscle quadrants. To understand how these dorsal-ventral differences in UNC-130 activity are established and maintained, we have analyzed the regulation of unc-130 expression and the distribution of UNC-130 protein. We have identified widespread, cis-acting elements in the unc-130 promoter that function to positively regulate ventral body wall muscle expression and negatively regulate dorsal body wall muscle expression. We have defined the temporal distribution of UNC-130 protein in body wall muscle cells during embryogenesis, demonstrated that this pattern is required to establish the dorsal-ventral polarity of UNC-129/TGF-β, and shown that UNC-130 is not required post-embryonically to maintain the asymmetry of body wall muscle unc-129 expression. Finally, we have tested the impact of the depletion of a variety of transcription factors, repressors, and signaling molecules to identify additional regulators of body wall muscle UNC-130 polarity. Our results confirm and extend earlier studies to clarify the mechanisms by which UNC-130 is controlled and affects the pattern of unc-129 expression in body wall muscle. These results further our understanding of the transcriptional logic behind the generation of polarity cues involving this poorly understood subclass of Forkhead factors.

The combinatorial expression of Hox genes along the body axes is a major determinant of cell fate and plays a pivotal role in generating the animal body plan. Loss of HOXA13 and HOXD13 transcription factors (HOX13) leads to digit agenesis in mice, but how HOX13 proteins regulate transcriptional outcomes and confer identity to the distal-most limb cells has remained elusive. Here, we report on the genome-wide profiling of HOXA13 and HOXD13 in vivo binding and changes of the transcriptome and chromatin state in the transition from the early to the late-distal limb developmental program, as well as in Hoxa13-/-; Hoxd13-/- limbs. Our results show that proper termination of the early limb transcriptional program and activation of the late-distal limb program are coordinated by the dual action of HOX13 on cis-regulatory modules.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.