Digit Identity Research Articles

Current research on mechanisms of limb bud development, and challenges for the next decade.

The developmental mechanisms of limb buds have been studied in developmental biology as an excellent model of pattern formation. Chick embryos have contributed to the discovery of new principles in developmental biology, as it is easy to observe live embryos and manipulate embryonic tissues. Herein, I outline recent findings and future issues over the next decade regarding three themes, based on my research: limb positioning, proximal-distal limb elongation and digit identity determination. First, how hindlimb position is determined at the molecular level is described, with a focus on the transforming growth factor-β signaling molecule GDF11. Second, I explain how the cell population in the limb bud deforms with developmental progress, shaping the limb bud with elongation along the proximal-distal axis. Finally, I describe the developmental mechanisms that determine digit identity through the interdigits.

Hippo signaling activates hedgehog signaling by Taz-driven Gli3 processing

The overlapping roles of Hippo and Hedgehog signaling in biological functions and diseases prompt us to investigate their potential interactions. Activation of Hippo signaling enhances the transcriptional output of Hedgehog signaling, and the role of Hippo signaling in regulating Hedgehog signaling relies on the Hippo pathway key effector, Taz. Interestingly, Taz exhibits a gradient expression across the posterior-to-anterior of limb bud mesoderms, similar to Sonic hedgehog (Shh). Importantly, Taz drives PKA to phosphorylate Gli3, resulting in the Gli3 processing into its repressor and attenuation of Hedgehog signaling in the Shh-independent manner. Specifically, Taz deletion in mouse embryonic limb bud mesenchyme not only enhances the Hedgehog signaling but partially restores the phenotypes from Shh deletion in causing severe defects of anteroposterior patterning and digit number and identity. Together, these results uncover Taz-dependent Gli3 processing as a hitherto uncharacterized mechanism controlling Hedgehog signaling, highlighting its cross-regulation by Hippo signaling.

The temporal dynamics of selective attention are reflected by distractor intrusions

When observers have to identify an object embedded in a rapid serial visual presentation (RSVP) stream, they often erroneously report the identity of a distractor instead of the target (distractor intrusion). In two experiments, we examined whether these intrusion errors are associated with the speed of attentional engagement. Participants reported the identity of target digits indicated by shape selection cues. To manipulate the speed of engagement, targets appeared either within a single RSVP stream or unpredictably in one of two streams. Objects that followed the selection cue were reported more frequently when engagement was delayed (two streams), whereas the probability of reporting objects preceding the cue was higher when engagement was faster (single stream). These results show that distractor intrusions are closely linked to the allocation of selective attention in time, making the intrusion paradigm a useful tool for research into the temporal dynamics of attention. They also provide new evidence for the idea that attentional selectivity operates within brief periods of perceptual enhancement (attentional episodes), facilitating the processing of all objects within this period, regardless of their status as targets or distractors.

Sonic hedgehog is not a limb morphogen but acts as a trigger to specify all digits in mice.

Limb patterning by Sonic hedgehog (Shh), via either graded spatial or temporal signal integration, is a paradigm for "morphogen" function, yet how Shh instructs distinct digit identities remains controversial. Here, we bypass the Shh requirement in cell survival during outgrowth and demonstrate that a transient, early Shh pulse is both necessary and sufficient for normal mouse limb development. Shh response is only short range and is limited to the Shh-expressing zone during this time window. Shh patterns digits 1-3, anterior to this zone, by an indirect mechanism rather than direct spatial or temporal signal integration. Using a genetic relay-signaling assay, we discover that Shh also specifies digit 1/thumb (thought to be exclusively Shh independent) indirectly, and this finding implicates Shh in a unique regulatory hierarchy for digit 1 evolutionary adaptations such as opposable thumbs. This study illuminates Shh as a trigger for an indirect downstream network that becomes rapidly self-sustaining, with mechanistic relevance for limb development, regeneration, and evolution.

Rethinking positional information and digit identity: The role of late interdigit signaling.

Seminal work from John Fallon's lab has illuminated how digit identity determination involves ongoing late regulation and occurs progressively during phalanx formation. Complementary genetic analyses in mice and several papers in this special issue have begun to flesh out how interdigit signaling accomplishes this, but major questions remain unaddressed, including how uncommitted progenitors from which phalanges arise are maintained, and what factors set limits on digit extension and phalanx number, particularly in mammals. This review summarizes what has been learned in the two decades since control of digit identity by late interdigit signals was first identified and what remains poorly understood, and will hopefully spark renewed interest in a process that is critical to evolutionary limb adaptations but nevertheless remains enigmatic.

The dynamic spatial and temporal relationships between the phalanx-forming region and the interdigits determine digit identity in the chick limb autopod.

Interdigits (IDs) determine digit identity in chick limbs. They are located between the digital rays and act as secondary signaling centers downstream of sonic hedgehog to provide positional information for determining digit identity in the phalanx-forming region (PFR). We examined the dynamic developmental mechanism by which PFR cells obtain positional information from IDs to determine the identity of individual digits in the chick hindlimb. We identified the specific region of the IDs responsible for determining digit identity and showed that PFR cells actively receive positional information only from the posteriorly, and not the anteriorly, located IDs. We also demonstrated that digits 1, 2, and 3 are interchangeable with each other, but not with digit 4. Finally, we found that both ID4 and digital ray 4 are necessary for determining digit 4 identity. The digital rays are naïve during the initial stages of their development, at which time digit identity is not determined. To determine digit identity, each PFR cell shows a unidirectional response to obtain positional information specifically from the IDs located posterior to the PFR, regardless of the signal strength from the anteriorly located IDs.

Reversibility of digit loss revisited: Limb diversification in Bachia lizards (gymnophthalmidae).

Strict interpretations of the Dollo's Law lead to postulation that trait loss is irreversible and organisms never recover ancestral phenotypes. Dollo, however, admitted the possibility of reversals in trait loss when predicted differences between reversed (derived) and ancestral forms. Phenotypic signatures from reversals are expected, as the historical context of a reversal in trait loss differs from the initial setting where the trait originally evolved. This article combines morphological and molecular information for Bachia scolecoides to discuss phenotypic and genetic patterns established during processes that reversed digit loss in Gymnophthalmidae (also termed microteiid lizards). Results suggest that pathways leading to the derived tetradactyl state of B. scolecoides comprise particularities in their origin and associated processes. Autopodial bones of B. scolecoides lack digit identity, and muscle anatomy is very similar between manus and pes. Gymnophthalmidae sequence patterns in the limb-specific sonic hedgehog enhancer (ZRS) suggest that regulation of shh expression did not degenerate in Bachia, given the prediction of similar motifs despite mutations specific to Bachia. Persistence of developmental mechanisms might explain intermittent character expression leading to reversals of digit loss, as ZRS signaling pathways remain active during the development of at least one pair of appendices in Bachia, especially if some precursors persisted at early stages. Patterns of ZRS sequences suggest that irreversibility of trait loss might be lineage-specific (restricted to Gymnophthalmini) and contingent to the type of signature established. These results provide insights regarding possible mechanisms that may allow reactivation of developmental programs in specific regions of the embryo.

Digit identity matters: origin and evolution of sexual dimorphism in the digit lengths of tropidurid lizards

AbstractSexual dimorphism in digit lengths reflects phenotypic evolution mediated by developmental steroids. Differences in the identity of the sexually-dimorphic digit may evolve if the concentrations of sex-steroid receptors in the digit are easily modified and the initial changes have low impact on fitness. Accordingly, sexual dimorphism in digit lengths might initially originate under neutral selective regimes, being subsequently co-opted by embryonic hormonal effects on sensitive traits that are more likely to be targeted by selection. Correlated variation among sexually-dimorphic traits might therefore reflect pleiotropic hormonal modulation during development. Moreover, the identity and trend of the sexually-dimorphic digit might be evolutionarily labile even among closely-related species. We evaluated this model by assessing the identity of sexually-dimorphic digits among 11 Tropiduridae lizard species and inferring evolutionary patterns of sexual dimorphism. Assuming that digit lengths can be used as a proxy for hormonal modulation of steroid-sensitive traits during development, we tested for evolutionary associations among sexual dimorphism of digit lengths, body size and other traits that may be direct targets of sexual selection in the Tropiduridae. Sexual dimorphism in digit lengths is evolutionarily labile in the Tropiduridae, and diversity, instead of conservatism, seems to rule developmental mechanisms underlying the evolution of sexual dimorphism in digit ratios.

An autoregulatory cell cycle timer integrates growth and specification in chick wing digit development.

A fundamental question is how proliferation and growth are timed during embryogenesis. Although it has been suggested that the cell cycle could be a timer, the underlying mechanisms remain elusive. Here we describe a cell cycle timer that operates in Sonic hedgehog (Shh)-expressing polarising region cells of the chick wing bud. Our data are consistent with Shh signalling stimulating polarising region cell proliferation via Cyclin D2, and then inhibiting proliferation via a Bmp2-p27kip1 pathway. When Shh signalling is blocked, polarising region cells over-proliferate and form an additional digit, which can be prevented by applying Bmp2 or by inhibiting D cyclin activity. In addition, Bmp2 also restores posterior digit identity in the absence of Shh signalling, thus indicating that it specifies antero-posterior (thumb to little finger) positional values. Our results reveal how an autoregulatory cell cycle timer integrates growth and specification and are widely applicable to many tissues.

Tbx5 inhibits hedgehog signaling in determination of digit identity.

Dominant TBX5 mutation causes Holt-Oram syndrome (HOS), which is characterized by limb defects in humans, but the underlying mechanistic basis is unclear. We used a mouse model with Tbx5 conditional knockdown in Hh-receiving cells (marked by Gli1+) during E8 to E10.5, a previously established model to study atrial septum defects, which displayed polydactyly or hypodactyly. The results suggested that Tbx5 is required for digit identity in a subset of limb mesenchymal cells. Specifically, Tbx5 deletion in this cell population decreased cell apoptosis and increased the proliferation of handplate mesenchymal cells. Furthermore, Tbx5 was found to negatively regulate the Hh-signaling activity through transcriptional regulation of Ptch1, a known Hh-signaling repressor. Repression of Hh-signaling through Smo co-mutation in Tbx5 heterozygotes rescued the limb defects, thus placing Tbx5 upstream of Hh-signaling in limb defects. This work reveals an important missing component necessary for understanding not only limb development but also the molecular and genetic mechanisms underlying HOS.

Evidence against tetrapod-wide digit identities and for a limited frame shift in bird wings

In crown group tetrapods, individual digits are homologized in relation to a pentadactyl ground plan. However, testing hypotheses of digit homology is challenging because it is unclear whether digits represent distinct and conserved gene regulatory states. Here we show dramatic evolutionary dynamism in the gene expression profiles of digits, challenging the notion that five digits have conserved developmental identities across amniotes. Transcriptomics shows diversity in the patterns of gene expression differentiation of digits, although the anterior-most digit of the pentadactyl limb has a unique, conserved expression profile. Further, we identify a core set of transcription factors that are differentially expressed among the digits of amniote limbs; their spatial expression domains, however, vary between species. In light of these results, we reevaluate the frame shift hypothesis of avian wing evolution and conclude only the identity of the anterior-most digit has shifted position, suggesting a 1,3,4 digit identity in the bird wing.

Limited-capacity identity processing of multiple integers.

The architecture of the numerical cognition system is currently not well understood, but at a general level, assumptions are made about two core components: a quantity processor and an identity processor. The quantity processor is concerned with accessing and using the stored magnitude denoted by a given digit, and the identity processor is concerned with recovery of the corresponding digit's identity. Blanc-Goldhammer and Cohen (Journal of Experimental Psychology: Learning, Memory, and Cognition, 40, 1389-1403, 2014) established that the recovery and use of quantity information operates in an unlimited-capacity fashion. Here we assessed whether the identity processor operates in a similar fashion. We present two experiments that were digit identity variations of Blanc-Goldhammer and Cohen's magnitude estimation paradigm. The data across both experiments reveal a limited-capacity identity processor whose operation reflects cross-talk with the quantity processor. Such findings provide useful evidence that can be used to adjudicate between competing models of the human number-processing system.

Are Face-Incongruent Voices Harder to Process?

Based on current integration theories of face-voice processing, the present study had participants process 1,152 videos of faces uttering digits. Half of the videos contained face-voice gender-incongruent stimuli (vs. congruent stimuli in the other half). Participants indicated digit magnitude or parity. Tasks were presented in pure blocks (only 1 task) and in task switching blocks (using colored cues to specify task). The results indicate significant congruency effects in pure blocks, but partially reversed congruency effects in task switching, probably due to enhanced assignment of capacity toward resolving difficult situational demands. Congruency effects did not dissipate over time, ruling out that initial surprise associated with incongruent stimuli drove the effects. The results show that interference between two task-irrelevant person-related dimensions (face/voice gender) can affect processing of a third, task-relevant dimension (digit identity), suggesting greater processing ease associated with more authentic voices (i.e., voices that do not violate face-based expectancies).

Does seeing gesture lighten or increase the load?

Abstract We examined the cognitive resources involved in processing speech with gesture compared to the same speech without gesture across four studies using a dual-task paradigm. Participants viewed videos of a woman describing spatial arrays either with gesture or without. They then attempted to choose the target array from among four choices. Participants’ cognitive load was measured as they completed this comprehension task by measuring how well they could remember the location and identity of digits in a secondary task. We found that addressees experience additional visuospatial load when processing gestures compared to speech alone, and that the load primarily comes when addressees attempt to use their memory of the descriptions with gesture to choose the target array. However, this cost only occurs when gestures about horizontal spatial relations (i.e., left and right) are produced from the speaker’s egocentric perspective.

Genetic regulatory pathways of split-hand/foot malformation.

Split-hand/foot malformation (SHFM) is caused by mutations in TP63, DLX5, DLX6, FGF8, FGFR1, WNT10B, and BHLHA9. The clinical features of SHFM caused by mutations of these genes are not distinguishable. This implies that in normal situations these SHFM-associated genes share an underlying regulatory pathway that is involved in the development of the central parts of the hands and feet. The mutations in SHFM-related genes lead to dysregulation of Fgf8 in the central portion of the apical ectodermal ridge (AER) and subsequently lead to misexpression of a number of downstream target genes, failure of stratification of the AER, and thus SHFM. Syndactyly of the remaining digits is most likely the effects of dysregulation of Fgf-Bmp-Msx signaling on apoptotic cell death. Loss of digit identity in SHFM is hypothesized to be the effects of misexpression of HOX genes, abnormal SHH gradient, or the loss of balance between GLI3A and GLI3R. Disruption of canonical and non-canonical Wnt signaling is involved in the pathogenesis of SHFM. Whatever the causative genes of SHFM are, the mutations seem to lead to dysregulation of Fgf8 in AER cells of the central parts of the hands and feet and disruption of Wnt-Bmp-Fgf signaling pathways in AER.

Developmental biology of the upper limb

This article aims to provide hand surgeons with current knowledge on the developmental biology of the upper limb. It will review positioning, limb bud emergence and formation of the apical ectodermal ridge. The development of the limb bud is analyzed in its three axes: proximal-distal, anteroposterior and dorsoventral. The signaling center and primary morphogens that initiate and stimulate the development of each axis will be described. For the proximal-distal axis, the apical ectodermal ridge stimulates the production of FGFs in the underlying distal mesoderm. The anteroposterior (or radio-ulnar) differentiation is a function of the zone of polarizing activity via the small Sonic hedgehog protein, which diffuses in a decreasing concentration gradient from the ulnar to the radial side of the bud. This gradient is essential to digit identity and numbers. For the dorsoventral differentiation, the signaling center is the dorsal ectoderm, which secretes WNT7A. Limb segmentation is described in three parts (arm, forearm and hand) along with the formation of the digital rays until finger separation. An example of congenital anomalies is provided for each step. To keep the length of this lecture within reason, the embryogenesis of nerves, blood vessels, muscles and tendons will not be discussed. On the other hand, the singularity of the thumb relative to the other fingers will be described. With a better understanding of developmental biology, surgeons should have better insight into congenital anomalies of the upper limb. This approach is the basis for the new OMT classification used by the IFFSH.

A solution to the problem of proper segment positioning in the course of digit formation

Digit formation requires a robust process. Both digit number and digit identity are precisely controlled. Alan Turing and Lewis Wolpert proposed two different mathematical processes for digit formation, yet neither of them is completely satisfactory. In this study, a process called “French-flag driven Turing patterning” is presented that takes the advantages of both previous systems. Four components have been considered: I) a short-range activator (diffusion factor), II.) a long-range inhibitor (diffusion factor) and III) a very-long acting diffusing inhibitor or “French-flag factor”, as well as IV) a non-diffusing, cell-autonomous transcription factor. The latter is responsible for the production of the activator and the long-range inhibitor. The “French-flag factor” is produced at one end of the one dimensional domain but its production decreases with time allowing the formation of the Turing pattern. Thus, the system becomes fast and extremely robust; peaks are formed always at the same position and it can support even 20% differences in the size of domains. The experimental findings derived from the examination of digit patterning are in full agreement with the results of the theoretical model. Although less experimental works are available, the presented “French-flag driven Turing patterning” mechanism might be a universal mechanism how nature produces highly reliable developmental segments.

Forward Genetics, Cilia, and Limb Development

Our research is focused on identifying genes and characterizing biological mechanisms that regulate organ formation and patterning during embryonic development. To identify key developmental and disease genes, we use powerful and unbiased forward genetics screens, which have become feasible due to recent advances in high-throughput sequencing. In the course of our studies, we have identified three mouse lines with mutations in cilia genes (Tmem107, Ttc26, Mks1). Cilia are microtubule-based, membrane-enclosed organelles found on the surface of almost all vertebrate cells and have been identified as key regulators of animal development and disease. The establishment of distinct digit identities is essential for the proper functioning of the limb and Sonic Hedgehog (Shh) signaling plays the major role in specifying the number and identity of digits. Recently, Shh signaling in vertebrates has been shown to be dependent upon the cilium. First, Shh pathway components including the receptor Ptch1, the positive pathway mediator Smo, and even the transcriptional mediators of the pathway, the Gli proteins, localize to the cilium. Second, mutations in genes that are required for cilia formation or maintenance cause Shh signaling defects in the embryo. Within the limb, cells with defective cilia generally do not perceive the Shh signal and lose the expression of genes downstream of Shh signaling. Surprisingly, the limbs of mouse cilia mutants do not resemble a Shh loss of function phenotype, but instead develop extra digits, i.e. polydactyly. Work from several labs has shown that cilia are required to form functional Gli-Activator (GliA). Cilia are also required to generate the processed repressor (GliR) forms, indicating that cilia are required for both the activator and repressor activities of Gli proteins. Loss of cilia or abnormal cilia function often results in polydactyly in mouse models and polydactyly is a defining feature of many cilia-related diseases (ciliopathies). We found that our cilia mutants (Tmem107, Ttc26, Mks1) appear to specifically affect the function of GliR in the limb. In addition, we have uncovered a late role for cilia in the regulation of programmed cell death in the limb. We are working to further define the genetic network that regulated cilia formation and function during limb development. Given the breadth and severity of cilia-associated defects, elucidating their underlying genetics is critical for diagnostics and defining the molecular function of cilia genes is essential to understanding how this small organelle impacts animal development and human health. Support or Funding Information R01AR059687 from NIAMS/NIH, P30DK090744 from NIDDK/NIH, training grant T32GM007499 from NIGMS/NIH, and training grant T32HD007149 from NICHD/NIH, and National Science Foundation Graduate Research Fellowships under DGE-0644492 and DGE-1122492. Disrupting cilia gene function causes polydactyly (asterisks). (A) Control, (B) Mks1 mutant, and (C) Tmem107 mutant E18.5 hindlimbs. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Digit identity influences numerical estimation in children and adults.

Learning the meanings of Arabic numerals involves mapping the number symbols to mental representations of their corresponding, approximate numerical quantities. It is often assumed that performance on numerical tasks, such as number line estimation (NLE), is primarily driven by translating from a presented numeral to a mental representation of its overall magnitude. Part of this assumption is that the overall numerical magnitude of the presented numeral, not the specific digits that comprise it, is what matters for task performance. Here we ask whether the magnitudes of the presented target numerals drive symbolic number line performance, or whether specific digits influence estimates. If the former is true, estimates of numerals with very similar magnitudes but different hundreds digits (such as 399 and 402) should be placed in similar locations. However, if the latter is true, these placements will differ significantly. In two studies (N = 262), children aged 7-11 and adults completed 0-1000 NLE tasks with target values drawn from a set of paired numerals that fell on either side of "Hundreds" boundaries (e.g., 698 and 701) and "Fifties" boundaries (e.g., 749 and 752). Study 1 used an atypical speeded NLE task, while Study 2 used a standard non-speeded NLE task. Under both speeded and non-speeded conditions, specific hundreds digits in the target numerals exerted a strong influence on estimates, with large effect sizes at all ages, showing that the magnitudes of target numerals are not the primary influence shaping children's or adults' placements. We discuss patterns of developmental change and individual difference revealed by planned and exploratory analyses.

Early limb patterning in the direct‐developing salamander Plethodon cinereus revealed by sox9 and col2a1

Direct-developing amphibians form limbs during early embryonic stages, as opposed to the later, often postembryonic limb formation of metamorphosing species. Limb patterning is dramatically altered in direct-developing frogs, but little attention has been given to direct-developing salamanders. We use expression patterns of two genes, sox9 and col2a1, to assess skeletal patterning during embryonic limb development in the direct-developing salamander Plethodon cinereus. Limb patterning in P. cinereus partially resembles that described in other urodele species, with early formation of digit II and a generally anterior-to-posterior formation of preaxial digits. Unlike other salamanders described to date, differentiation of preaxial zeugopodial cartilages (radius/tibia) is not accelerated in relation to the postaxial cartilages, and there is no early differentiation of autopodial elements in relation to more proximal cartilages. Instead, digit II forms in continuity with the ulnar/fibular arch. This amniote-like connectivity to the first digit that forms may be a consequence of the embryonic formation of limbs in this direct-developing species. Additionally, and contrary to recent models of amphibian digit identity, there is no evidence of vestigial digits. This is the first account of gene expression in a plethodontid salamander and only the second published account of embryonic limb patterning in a direct-developing salamander species.