Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

The limb dorsoventral axis: Lmx1b's role in development, pathology, evolution, and regeneration.

The limb anatomy displays well-defined dorsal and ventral compartments, housing extensor, and flexor muscles, which play a crucial role in facilitating limb locomotion and manipulation. Despite its importance, the study of limb dorsoventral patterning has been relatively neglected compared to the other two axes leaving many crucial questions about the genes and developmental processes implicated unanswered. This review offers a thorough overview of the current understanding of limb dorsoventral patterning, synthesizing classical literature with recent research. It covers the specification of dorsal fate in the limb mesoderm and its subsequent translation into dorsal morphologies-a process directed by the transcription factor Lmx1b. We also discuss the potential role of dorsoventral patterning in the evolution of paired appendages and delve into the involvement of LMX1B in Nail-Patella syndrome, discussing the molecular and genetic aspects underlying this condition. Finally, the potential role of dorsoventral polarity in digit tip regeneration, a prominent instance of multi-tissue regeneration in mammals is also considered. We anticipate that this review will renew interest in a process that is critical to limb function and evolutionary adaptations but has nonetheless been overlooked.

Failure of digit tip regeneration in the absence of Lmx1b suggests Lmx1b functions disparate from dorsoventral polarity.

Mammalian digit tip regeneration is linked to the presence of nail tissue, but a nail-explicit model is missing. Here, we report that nail-less double-ventral digits of ΔLARM1/2 mutants that lack limb-specific Lmx1b enhancers fail to regenerate. To separate the nail's effect from the lack of dorsoventral (DV) polarity, we also interrogate double-dorsal double-nail digits and show that they regenerate. Thus, DV polarity is not a prerequisite for regeneration, and the nail requirement is supported. Transcriptomic comparison between wild-type and non-regenerative ΔLARM1/2 mutant blastemas reveals differential upregulation of vascularization and connective tissue functional signatures in wild type versus upregulation of inflammation in the mutant. These results, together with the finding of Lmx1b expression in the postnatal dorsal dermis underneath the nail and uniformly in the regenerative blastema, open the possibility of additional Lmx1b roles in digit tip regeneration, in addition to the indirect effect of mediating the formation of the nail.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

Downregulation of Grem1 expression in the distal limb mesoderm is a necessary precondition for phalanx development.

BACKGROUND: The phalanges are the final skeletal elements to form in the vertebrate limb and their identity is regulated by signaling at the phalanx forming region (PFR) located at the tip of the developing digit ray. Here, we seek to explore the relationship between PFR activity and phalanx morphogenesis, which define the most distal limb skeletal elements, and signals associated with termination of limb outgrowth. RESULTS: As Grem1 is extinguished in the distal chick limb mesoderm, the chondrogenesis marker Aggrecan is up-regulated in the metatarsals and phalanges. Fate mapping confirms that subridge mesoderm cells contribute to the metatarsal and phalanges when subridge Grem1 is down-regulated. Grem1 overexpression specifically blocks chick phalanx development by inhibiting PFR activity. PFR activity and digit development are also disrupted following overexpression of a Gli3 repressor, which results in Grem1 expression in the distal limb and downregulation of Bmpr1b. CONCLUSIONS: Based on expression and fate mapping studies, we propose that downregulation of Grem1 in the distal limb marks the transition from metatarsal to phalanx development. This suggests that downregulation of Grem1 in the distal limb mesoderm is necessary for phalanx development. Grem1 downregulation allows for full PFR activity and phalanx progenitor cell commitment to digit fate.

Time-sequenced transcriptomes of developing distal mouse limb buds: A comparative tissue layer analysis.

BACKGROUND: The development of the amniote limb has been an important model system to study patterning mechanisms and morphogenesis. For proper growth and patterning, it requires the interaction between the distal sub-apical mesenchyme and the apical ectodermal ridge (AER) that involve the separate implementation of coordinated and tissue-specific genetic programs. RESULTS: Here, we produce and analyze the transcriptomes of both distal limb mesenchymal progenitors and the overlying ectodermal cells, following time-coursed dissections that cover from limb bud initiation to fully patterned limbs. The comparison of transcriptomes within each layer as well as between layers over time, allowed the identification of specific transcriptional signatures for each of the developmental stages. Special attention was given to the identification of genes whose transcription dynamics suggest a previously unnoticed role in the context of limb development and also to signaling pathways enriched between layers. CONCLUSION: We interpret the transcriptomic data in light of the known development pattern and we conclude that a major transcriptional transition occurs in distal limb buds between E9.5 and E10.5, coincident with the switch from an early phase continuation of the signature of trunk progenitors, related to the initial proximo distal specification, to a late intrinsic phase of development.

Identification of limb-specific Lmx1b auto-regulatory modules with Nail-patella syndrome pathogenicity.

LMX1B haploinsufficiency causes Nail-patella syndrome (NPS; MIM 161200), characterized by nail dysplasia, absent/hypoplastic patellae, chronic kidney disease, and glaucoma. Accordingly in mice, Lmx1b has been shown to play crucial roles in the development of the limb, kidney and eye. Although one functional allele of Lmx1b appears adequate for development, Lmx1b null mice display ventral-ventral distal limbs with abnormal kidney, eye and cerebellar development, more disruptive, but fully concordant with NPS. In Lmx1b functional knockouts (KOs), Lmx1b transcription in the limb is decreased nearly 6-fold, indicating autoregulation. Herein, we report on two conserved Lmx1b-associated cis-regulatory modules (LARM1 and LARM2) that are bound by Lmx1b, amplify Lmx1b expression with unique spatial modularity in the limb, and are necessary for Lmx1b-mediated limb dorsalization. These enhancers, being conserved across vertebrates (including coelacanth, but not other fish species), and required for normal locomotion, provide a unique opportunity to study the role of dorsalization in the fin to limb transition. We also report on two NPS patient families with normal LMX1B coding sequence, but with loss-of-function variations in the LARM1/2 region, stressing the role of regulatory modules in disease pathogenesis.

Mammalian-specific ectodermal enhancers control the expression of Hoxc genes in developing nails and hair follicles.

Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.

The formation of the thumb requires direct modulation of Gli3 transcription by Hoxa13.

In the tetrapod limb, the digits (fingers or toes) are the elements most subject to morphological diversification in response to functional adaptations. However, despite their functional importance, the mechanisms controlling digit morphology remain poorly understood. Here we have focused on understanding the special morphology of the thumb (digit 1), the acquisition of which was an important adaptation of the human hand. To this end, we have studied the limbs of the Hoxa13 mouse mutant that specifically fail to form digit 1. We show that, consistent with the role of Hoxa13 in Hoxd transcriptional regulation, the expression of Hoxd13 in Hoxa13 mutant limbs does not extend into the presumptive digit 1 territory, which is therefore devoid of distal Hox transcripts, a circumstance that can explain its agenesis. The loss of Hoxd13 expression, exclusively in digit 1 territory, correlates with increased Gli3 repressor activity, a Hoxd negative regulator, resulting from increased Gli3 transcription that, in turn, is due to the release from the negative modulation exerted by Hox13 paralogs on Gli3 regulatory sequences. Our results indicate that Hoxa13 acts hierarchically to initiate the formation of digit 1 by reducing Gli3 transcription and by enabling expansion of the 5'Hoxd second expression phase, thereby establishing anterior-posterior asymmetry in the handplate. Our work uncovers a mutual antagonism between Gli3 and Hox13 paralogs that has important implications for Hox and Gli3 gene regulation in the context of development and evolution.

An intrinsic cell cycle timer terminates limb bud outgrowth.

The longstanding view of how proliferative outgrowth terminates following the patterning phase of limb development involves the breakdown of reciprocal extrinsic signalling between the distal mesenchyme and the overlying epithelium (e-m signalling). However, by grafting distal mesenchyme cells from late stage chick wing buds to the epithelial environment of younger wing buds, we show that this mechanism is not required. RNA sequencing reveals that distal mesenchyme cells complete proliferative outgrowth by an intrinsic cell cycle timer in the presence of e-m signalling. In this process, e-m signalling is required permissively to allow the intrinsic cell cycle timer to run its course. We provide evidence that a temporal switch from BMP antagonism to BMP signalling controls the intrinsic cell cycle timer during limb outgrowth. Our findings have general implications for other patterning systems in which extrinsic signals and intrinsic timers are integrated.

Systematic analysis of transcription start sites in avian development.

Cap Analysis of Gene Expression (CAGE) in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs) and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM]) were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals). By facilitating promoter-based molecular analysis and genetic manipulation, our work also underscores the value of avian models in unravelling the complex regulatory mechanism of cell lineage specification during amniote development.

HOX13 proteins: the molecular switcher in Hoxd bimodal regulation.

The striking correlation between the genomic arrangement of Hox genes and their temporal and spatial pattern of expression during embryonic development has been a source of fascination since its discovery. This correspondence has been used as a privileged example in the investigation of the connection between genomic architecture and function. In this issue of Genes & Development, Beccari and colleagues (pp. 1172-1186) make a big step forward in understanding Hox gene regulation during limb development by showing the pivotal role of HOXA13 and HOXD13 proteins in the transition from a proximal to a distal type of Hoxd transcriptional regulation.

Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development.

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/ßcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.

Long bone development requires a threshold of Hox function.

The Hoxd(Del(11-13)) mutant is one of the animal models for human synpolydactyly, characterized by short and syndactylous digits. Here we have characterized in detail the cartilage and bone defects in these mutants. We report two distinct phenotypes: (i) a delay and change in pattern of chondrocyte maturation of metacarpals/metatarsals and (ii) formation of a poor and not centrally positioned primary ossification center in the proximal-intermediate phalanx. In the metacarpals of Hoxd(Del(11-13)) mutants, ossification occurs postnataly, in the absence of significant Ihh expression and without the establishment of growth plates, following patterns similar to those of short bones. The strong downregulation in Ihh expression is associated with a corresponding increase of the repressor form of Gli3. To evaluate the contribution of this alteration to the phenotype, we generated double Hoxd(Del(11-13));Gli3 homozygous mutants. Intriguingly, these double mutants showed a complete rescue of the phenotype in metatarsals but only partial phenotypic rescue in metacarpals. Our results support Hox genes being required in a dose-dependent manner for long bone cartilage maturation and suggest that and excess of Gli3R mediates a significant part of the Hoxd(Del(11-13)) chondrogenic phenotype.

Decoupling the function of Hox and Shh in developing limb reveals multiple inputs of Hox genes on limb growth.

Limb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hox-dependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes.

How do we get a perfect complement of digits?

A crucial issue in limb development is how a correct set of precisely shaped digits forms in the digital plate. This process relies on patterning across the anterior-posterior axis of the limb bud, which is under the control of Sonic hedgehog emanating from the zone of polarizing activity. Recently, Sonic hedgehog function in the limb bud has been shown to have a dual character controlling both growth and patterning of the digital field. This finding has prompted the proposal of new models of how these two functions are achieved, and this will be discussed in this review.

Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling.

The bHLH transcription factor dHAND is required for establishment of SHH signaling by the limb bud organizer in posterior mesenchyme, a step crucial to development of vertebrate paired appendages. We show that the transcriptional repressor GLI3 restricts dHAND expression to posterior mesenchyme prior to activation of SHH signaling in mouse limb buds. dHAND, in turn, excludes anterior genes such as Gli3 and Alx4 from posterior mesenchyme. Furthermore, genetic interaction of GLI3 and dHAND directs establishment of the SHH/FGF signaling feedback loop by restricting the BMP antagonist GREMLIN posteriorly. These interactions polarize the nascent limb bud mesenchyme prior to SHH signaling.