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Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months 1 . This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common 2 . Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species. Using single-cell and spatial transcriptomics, human embryonic limb development across space and time and the diversification and cross-species conservation of cells are demonstrated.

Key Points Taste buds are composed of two excitable cell types and a glia-like cell; each type of cell has distinct functions. Basic taste qualities are detected by G protein-coupled type 1 and type 2 taste receptors, by other receptors and ion channels, and possibly by transporters. ATP is an afferent taste transmitter and is secreted by taste bud cells through an unconventional, non-vesicular release mechanism. ATP, serotonin and GABA mediate cell–cell interactions in the taste bud that may shape transmission to sensory afferent fibres. Controversy remains regarding whether peripheral taste coding follows a labelled-line or combinatorial pattern. Taste preferences and appetites seem to have a genetic component that is being revealed by molecular and population studies. Mammals detect the nutrient content, palatability and potential toxicity of food through taste buds that are present mainly in the tongue. In this Review, Roper and Chaudhari discuss the taste bud cells, receptors and transmitters that are involved in taste detection, how these cells communicate with sensory afferent fibres, and peripheral taste coding. The past decade has witnessed a consolidation and refinement of the extraordinary progress made in taste research. This Review describes recent advances in our understanding of taste receptors, taste buds, and the connections between taste buds and sensory afferent fibres. The article discusses new findings regarding the cellular mechanisms for detecting tastes, new data on the transmitters involved in taste processing and new studies that address longstanding arguments about taste coding.

Taste-receptor cells use distinct semaphorins to guide wiring of the peripheral taste system; targeted ectopic expression of SEMA3A or SEMA7A leads to bitter neurons responding to sweet tastes or sweet neurons responding to bitter tastes. Bittersweet tinkering with the taste system Taste cells experience a very rapid turnover, having life spans of only 5 to 20 days, but it is not yet known how the constantly replenishing taste cells re-establish appropriate connections with their respective ganglion neurons. Here, Charles Zuker and colleagues reveal that taste receptor cells make connections with neurons representing the same taste quality based on the different axon guidance molecules expressed by each taste receptor cell type. To demonstrate this molecular logic, the authors forced a sweet taste receptor cell to establish a connection with a bitter taste quality neuron simply through the ectopic expression of the bitter guidance molecule in the sweet taste receptor cell. These findings provide insights into how the gustatory system remains organized and specific despite experiencing cell turnover on such a large scale. In mammals, taste buds typically contain 50–100 tightly packed taste-receptor cells (TRCs), representing all five basic qualities: sweet, sour, bitter, salty and umami 1 , 2 . Notably, mature taste cells have life spans of only 5–20 days and, consequently, are constantly replenished by differentiation of taste stem cells 3 . Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (that is, ensuring that a labelled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, and so on), we examined how new connections are specified to retain fidelity of signal transmission. Here we show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (SEMA3A and SEMA7A) 4 , 5 , 6 . We demonstrate that targeted expression of SEMA3A or SEMA7A in different classes of TRCs produces peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered mice with bitter neurons that now responded to sweet tastants, sweet neurons that responded to bitter or sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labelled-line sensory circuit preserves signalling integrity despite rapid and stochastic turnover of receptor cells.

High concentrations of salt activate sour- and bitter-taste-sensing cells in the tongues of mice, and genetic silencing of these pathways abolishes behavioural aversion to concentrated salt; this ‘co-opting’ of the two primary aversive taste pathways (sour and bitter) may have evolved so that high salt levels reliably trigger behavioural rejection. The taste of too much salt In contrast to the other four basic tastes (sweet, umami, sour and bitter), which are either appetitive or aversive, sodium salt can be both attractive and repulsive, depending on concentration. Lower concentrations of salt are perceived by cells expressing the sodium channel ENaC. In this study, Charles Zuker and colleagues show that high levels of salt activate the sour and bitter taste-sensing cells, and that salt-avoidance behaviours are abolished in mice lacking these pathways. The authors conclude that 'co-opting' the two primary aversive taste pathways causes the animals to reject foodstuffs containing extreme — and potentially harmful — levels of salt. Given current concerns about excessive dietary salt intake in humans, this work raises the prospect of developing selective receptor-cell modulators to help to control or even satisfy our strong appetite for salt without the potential ill effects of too much sodium. In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami 1 , 2 . Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one 3 , 4 , 5 , 6 , 7 . This appetitive–aversive balance helps to maintain appropriate salt consumption 3 , 4 , 6 , 8 , and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC 8 , but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the ‘co-opting’ of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health.

The red light:far-red light ratio perceived by phytochromes controls plastic traits of plant architecture, including branching. Despite the significance of branching for plant fitness and productivity, there is little quantitative and mechanistic information concerning phytochrome control of branching responses in Arabidopsis (Arabidopsis thaliana). Here, we show that in Arabidopsis, the negative effects of the phytochrome B mutation and of low red light:far-red light ratio on branching were largely due to reduced bud outgrowth capacity and an increased degree of correlative inhibition acting on the buds rather than due to a reduced number of leaves and buds available for branching. Phytochrome effects on the degree of correlative inhibition required functional BRANCHED1 (BRC1), BRC2, AXR1, MORE AXILLARY GROWTH2 (MAX2), and MAX4. The analysis of gene expression in selected buds indicated that BRC1 and BRC2 are part of different gene networks. The BRC1 network is linked to the growth capacity of specific buds, while the BRC2 network is associated with coordination of growth among branches. We conclude that the branching integrators BRC1 and BRC2 are necessary for responses to phytochrome, but they contribute differentially to these responses, likely acting through divergent pathways.

Striking taste disturbances are reported in cancer patients treated with Hedgehog (HH)-pathway inhibitor drugs, including sonidegib (LDE225), which block the HH pathway effector Smoothened (SMO). We tested the potential for molecular, cellular, and functional recovery in mice from the severe disruption of taste-organ biology and taste sensation that follows HH/SMO signaling inhibition. Sonidegib treatment led to rapid loss of taste buds (TB) in both fungiform and circumvallate papillae, including disruption of TB progenitor-cell proliferation and differentiation. Effects were selective, sparing nontaste papillae. To confirm that taste-organ effects of sonidegib treatment result from HH/SMO signaling inhibition, we studied mice with conditional global or epithelium-specific Smo deletions and observed similar effects. During sonidegib treatment, chorda tympani nerve responses to lingual chemical stimulation were maintained at 10 d but were eliminated after 16 d, associated with nearly complete TB loss. Notably, responses to tactile or cold stimulus modalities were retained. Further, innervation, which was maintained in the papilla core throughout treatment, was not sufficient to sustain TB during HH/SMO inhibition. Importantly, treatment cessation led to rapid and complete restoration of taste responses within 14 d associated with morphologic recovery in about 55% of TB. However, although taste nerve responses were sustained, TB were not restored in all fungiform papillae even with prolonged recovery for several months. This study establishes a physiologic, selective requirement for HH/SMO signaling in taste homeostasis that includes potential for sensory restoration and can explain the temporal recovery after taste dysgeusia in patients treated with HH/SMO inhibitors.

• Resting bud cataphylls are often assumed to play an essential protective role in winter due to their widespread presence among temperate, woody plants. This view is challenged by our documentation of significant numbers of temperate woody angiosperm taxa with naked buds that overwinter without cataphyll protection. • We inventoried temperate, woody angiosperm taxa reported to have resting buds without cataphyll protection in winter and for the first time characterised the morphological and functional diversity of naked buds. Using this new classification of bud types, the taxonomic and geographic distributions of taxa with naked buds were summarised and relationships between plant functional traits and bud type were investigated. • Naked buds are not, as long presumed, markedly rare in temperate, woody floras. They occur in at least 87 genera in 42 families throughout the angiosperm phylogeny in various morphologically distinct manifestations. The geographic distribution of species with naked buds in temperate areas was found to be associated with summer precipitation, but not with winter climatic variables. • Resting bud structure is not necessarily a trait optimised solely for winter survival. A taxon’s bud composition may be influenced by factors such as biogeographic history and ontogenetic pattern of leaf formation over the growing season.

Action potentials play an important role in neurotransmitter release in response to taste. Here, I have investigated voltage-gated Na+ channels, a primary component of action potentials, in respective cell types of mouse fungiform taste bud cells (TBCs) with in situ whole-cell clamping and single-cell RT-PCR techniques. The cell types of TBCs electrophysiologically examined were determined immunohistochemically using the type III inositol 1,4,5-triphoshate receptor as a type II cell marker and synaptosomal-associated protein 25 as a type III cell marker. I show that type II cells, type III cells, and TBCs not immunoreactive to these markers (likely type I cells) generate voltage-gated Na+ currents. The recovery following inactivation of these currents was well fitted with double exponential curves. The time constants in type III cells (~20 ms and ~ 1 s) were significantly slower than respective time constants in other cell types. RT-PCR analysis indicated the expression of Nav1.3, Nav1.5, Nav1.6, and β1 subunit mRNAs in TBCs. Pharmacological inhibition and single-cell RT-PCR studies demonstrated that type II and type III cells principally express tetrodotoxin (TTX)-sensitive Nav1.3 channels and that ~ 30% of type I cells express TTX-resistant Nav1.5 channels. The auxiliary β1 subunit that modulates gating kinetics was rarely detected in TBCs. As the β1 subunit co-expressed with an α subunit is known to accelerate the recovery from inactivation, it is likely that voltage-gated Na+ channels in TBCs may function without β subunits. Slow recovery from inactivation, especially in type III cells, may limit high-frequency firing in response to taste substances.

The cellular interactions controlling digit numbers and identities have remained largely elusive. Here, we leverage the anterior digit and identity loss in Grem1 tetradactyl mouse limb buds to identify early specified limb bud mesenchymal progenitor (LMP) populations whose size and distribution is governed by spatial modulation of BMP activity and SHH signaling. D istal-autopodial LMPs (dLMP) express signature genes required for autopod and digit development, and alterations affecting the dLMP population size prefigure the changes in digit numbers that characterize specific congenital malformations. A second, p eripheral LMP (pLMP) population is anteriorly biased and reduction/loss of its asymmetric distribution underlies the loss of middle digit asymmetry and identities in Grem1 tetradactyl and pig limb buds. pLMPs depend on BMP activity, while dLMPs require GREM1-mediated BMP antagonism. Taken together, the spatial alterations in GREM1 antagonism in mouse mutant and evolutionarily diversified pig limb buds tunes BMP activity, which impacts dLMP and pLMP populations in an opposing manner. The initial cellular alterations underlying changes in digit numbers and identities were unknown. Here, Palacio et al. identify two limb bud progenitor populations that are impacted in an opposing manner by changes in BMP antagonism linked to congenital and evolutionary digit variations.