Developing Limb Bud Research Articles

Sall4 regulates downstream patterning genes during limb regeneration

Many salamanders can completely regenerate a fully functional limb. Limb regeneration is a carefully coordinated process involving several defined stages. One key event during the regeneration process is the patterning of the blastema to inform cells of what they must differentiate into. Although it is known that many genes involved in the initial development of the limb are re-used during regeneration, the exact molecular circuitry involved in this process is not fully understood. Several large-scale transcriptional profiling studies of axolotl limb regeneration have identified many transcription factors that are up-regulated after limb amputation. Sall4 is a transcription factor that has been identified to play essential roles in maintaining cells in an undifferentiated state during development and also plays a unique role in limb development. Inactivation of Sall4 during limb bud development results in defects in anterior-posterior patterning of the limb. Sall4 has been found to be up-regulated during limb regeneration in both Xenopus and salamanders, but to date it function has been untested. We confirmed that Sall4 is up-regulated during limb regeneration in the axolotl using qRT-PCR and identified that it is present in the skin cells and also in cells within the blastema. Using CRISPR technology we microinjected gRNAs specific for Sall4 complexed with cas9 protein into the blastema to specifically knockout Sall4 in blastema cells only. This resulted in limb regenerate defects, including missing digits, fusion of digit elements, and defects in the radius and ulna. This suggests that during regeneration Sall4 may play a similar role in regulating the specification of anterior-proximal skeletal elements.

TBX3 is essential for establishment of the posterior boundary of anterior genes and upregulation of posterior genes together with HAND2 during the onset of limb bud development.

During limb bud formation, axis polarities are established as evidenced by the spatially restricted expression of key regulator genes. In particular, the mutually antagonistic interaction between the GLI3 repressor and HAND2 results in distinct and non-overlapping anterior-distal Gli3 and posterior Hand2 expression domains. This is a hallmark of the establishment of antero-posterior limb axis polarity, together with spatially restricted expression of homeodomain and other transcriptional regulators. Here, we show that TBX3 is required for establishment of the posterior expression boundary of anterior genes in mouse limb buds. ChIP-seq and differential gene expression analysis of wild-type and mutant limb buds identifies TBX3-specific and shared TBX3-HAND2 target genes. High sensitivity fluorescent whole-mount in situ hybridisation shows that the posterior expression boundaries of anterior genes are positioned by TBX3-mediated repression, which excludes anterior genes such as Gli3, Alx4, Hand1 and Irx3/5 from the posterior limb bud mesenchyme. This exclusion delineates the posterior mesenchymal territory competent to establish the Shh-expressing limb bud organiser. In turn, HAND2 is required for Shh activation and cooperates with TBX3 to upregulate shared posterior identity target genes in early limb buds.

Loss of the ability to regenerate body appendages in vertebrates: from side effects of evolutionary innovations to gene loss.

The ability to regenerate large body appendages is an ancestral trait of vertebrates, which varies across different animal groups. While anamniotes (fish and amphibians) commonly possess this ability, it is notably restricted in amniotes (reptiles, birds, and mammals). In this review, we explore the factors contributing to the loss of regenerative capabilities in amniotes. First, we analyse the potential negative impacts on appendage regeneration caused by four evolutionary innovations: advanced immunity, skin keratinization, whole-body endothermy, and increased body size. These innovations emerged as amniotes transitioned to terrestrial habitats and were correlated with a decline in regeneration capability. Second, we examine the role played by the loss of regeneration-related enhancers and genes initiated by these innovations in the fixation of an inability to regenerate body appendages at the genomic level. We propose that following the cessation of regenerative capacity, the loss of highly specific regeneration enhancers could represent an evolutionarily neutral event. Consequently, the loss of such enhancers might promptly follow the suppression of regeneration as a side effect of evolutionary innovations. By contrast, the loss of regeneration-related genes, due to their pleiotropic functions, would only take place if such loss was accompanied by additional evolutionary innovations that compensated for the loss of pleiotropic functions unrelated to regeneration, which would remain even after participation of these genes in regeneration was lost. Through a review of the literature, we provide evidence that, in many cases, the loss in amniotes of genes associated with body appendage regeneration in anamniotes was significantly delayed relative to the time when regenerative capability was lost. We hypothesise that this delay may be attributed to the necessity for evolutionary restructuring of developmental mechanisms to create conditions where the loss of these genes was a beneficial innovation for the organism. Experimental investigation of the downregulation of genes involved in the regeneration of body appendages in anamniotes but absent in amniotes offers a promising avenue to uncover evolutionary innovations that emerged from the loss of these genes. We propose that the vast majority of regeneration-related genes lost in amniotes (about 150 in humans) may be involved in regulating the early stages of limb and tail regeneration in anamniotes. Disruption of this stage, rather than the late stage, may not interfere with the mechanisms of limb and tail bud development during embryogenesis, as these mechanisms share similarities with those operating in the late stage of regeneration. Consequently, the most promising approach to restoring regeneration in humans may involve creating analogs of embryonic limb buds using stem cell-based tissue-engineering methods, followed by their transfer to the amputation stump. Due to the loss of many genes required specifically during the early stage of regeneration, this approach may be more effective than attempting to induce both early and late stages of regeneration directly in the stump itself.

MSX1+PDGFRAlow limb mesenchyme-like cells as an efficient stem cell source for human cartilage regeneration

Degenerative bone disorders have a significant impact on global health, and regeneration of articular cartilage remains a challenge. Existing cell therapies using mesenchymal stromal cells (MSCs) have shown limited efficacy, highlighting the necessity for alternative stem cell sources. Here, we have identified and characterized MSX1+ mesenchymal progenitor cells in the developing limb bud with remarkable osteochondral-regenerative and microenvironment-adaptive capabilities. Single-cell sequencing further revealed the presence of two major cell compositions within the MSX1+ cells, where a distinct PDGFRAlow subset retained the strongest osteochondral competency and could efficiently regenerate articular cartilage invivo. Furthermore, a strategy was developed to generate MSX1+PDGFRAlow limb mesenchyme-like (LML) cells from human pluripotent stem cells that closely resembled their mouse counterparts, which were bipotential invitro and could directly regenerate damaged cartilage in a mouse injury model. Together, our results indicated that MSX1+PDGFRAlow LML cells might be a prominent stem cell source for human cartilage regeneration.

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.

The pre- and postembryonic development of Artemia franciscana (Anostraca: Artemiidae).

Artemia franciscana is a universal live feed in aquaculture, and it has been reported as an invasive species in many Asian hypersaline ecosystems. The present observations illustrated the pre- and postembryonic development stages of the A. franciscana population confined to the Indian saltern of Kelambakkam. We observed their growth patterns during various hydration periods with specific time intervals. Results showed differences in the development stages with respect to unique identity. Interestingly, a period of hydration showed notable cellular movement toward clockwise positions in the hydrating cysts. After 10 h of hydration, blastocoel appeared, accelerating the dynamic route of nuclei movement. At the end of the invagination, the embryo burst out of the cyst, and a sequence of emerging stages was noted. With reference to light microscopic observations, a series of developmental stages were observed, and each instar was documented by developing limb buds of nauplii. Excitingly, the 10th and 11th instar stages reveal sexual differentiation between male and female individuals. Thus, the laboratory culture study clearly documented the different developmental stages with their specific characteristic features. However, further molecular study would provide a cellular basis for understanding the early development of A. franciscana.

Hnrnpk is essential for embryonic limb bud development as a transcription activator and a collaborator of insulator protein Ctcf

Proper development of the limb bud relies on the concordance of various signals, but its molecular mechanisms have not yet been fully illustrated. Here we report that heterogeneous nuclear ribonucleoprotein K (hnRNPK) is essential for limb bud development. Its ablation in the limb bud results in limbless forelimbs and severe deformities of the hindlimbs. In terms of mechanism, hnRNPK functions as a transcription activator for the vital genes involved in the three regulatory axes of limb bud development. Simultaneously, for the first time we elucidate that hnRNPK binds to and coordinates with the insulator protein CCCTC binding factor (CTCF) to maintain a three-dimensional chromatin architecture. Ablation of hnRNPK weakens the binding strength of CTCF to topologically associating domain (TAD) boundaries, then leading to the loose TADs, and decreased interactions between promoters and enhancers, and further decreased transcription of developmental genes. Our study establishes a fundamental and novel role of hnRNPK in regulating limb bud development.

Innervation of the developing kidney in vivo and in vitro.

Within the adult kidney, renal neurites can be observed alongside the arteries where they play a role in regulating blood flow. However, their role and localization during development has so far not been described in detail. In other tissues, such as the skin of developing limb buds, neurons play an important role during arterial differentiation. Here, we aim to investigate whether renal nerves could potentially carry out a similar role during arterial development in the mouse kidney. In order to do so, we used whole-mount immunofluorescence staining to identify whether the timing of neuronal innervation correlates with the recruitment of arterial smooth muscle cells. Our results show that neurites innervate the kidney between day 13.5 and 14.5 of development, arriving after the recruitment of smooth muscle actin-positive cells to the renal arteries.

Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

Sall4 restricts glycolytic metabolism in limb buds through transcriptional regulation of glycolytic enzyme genes

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds.

Characterizing Pathogenic Enhancer Activity at Single-cell Resolution

The ZRS enhancer, a regulatory sequence found in numerous organisms, plays an important role in early embryonic limb development. ZRS controls the expression of the Sonic Hedgehog gene (Shh), and therefore early limb development in an organism as Shh has been shown to control the width of the limb bud by stimulating mesenchyme cell proliferation due to its ability to regulate the anterior-posterior length of the apical ectodermal ridge. Several transcription factors, acting as repressors or activators of the Shh gene, coordinate this limb development process in tandem with the ZRS enhancer. While the significance of normal ZRS activity is evident, this study looks deeper into the effects of pathogenic changes to the ZRS enhancer and the development of associated limb disorders such as preaxial polydactyly (PPD) by focusing on several aspects of ZRS regulation and its relation to Shh expression. This was accomplished by characterizing the expression of Shh and mCherry, an introduced luminescence gene regulated by ZRS, through single-cell RNA sequenced cells from a developing limb bud of a mouse embryo. Additionally, this study characterized specific transcription factors as potential repressors or activators of ZRS by determining TF enrichment or depletion in highly expressive Shh and mCherry cells. Classifying such TFs is vital in identifying the regulatory elements that control the formation of limb malformation disorders such as preaxial polydactyly and aid in the development of future therapeutic interventions.

A review of polydactyly and its inheritance: Connecting the dots

This study collects what is known about the inheritance underpinnings of syndromic and non-syndromic polydactylies and highlights dactyly presentations with unknown genetic roots. This review summarizes the current information and genetics-enhanced understanding of polydactyly. There is a frequency of 0.37 to 1.2 per 1000 live births for polydactyly, which is also known as hyperdactyly. It is characterized by the presence of extra fingers. Polydactyly is caused by a failure in limb development, specifically the patterning of the developing limb bud. The phenotypic and genetic variability of polydactyly makes its etiology difficult to understand. Pre-axial polydactyly, central polydactyly (axial), and postaxial polydactyly are all examples of non-syndromic polydactyly (ulnar). An autosomal dominant disorder with varying penetrance that is mostly passed down via limb development patterning abnormalities. A comprehensive search of MEDLINE/PubMed and other databases was followed by an evaluation of the relevant papers, with a particular focus on those published between 2000 and 2022. Of 747 published article related to Polydactyly from MEDLINE/PubMed search, 43 were from the last 10 years and were the focus of this review. Polydactyly is one of the most frequent congenital hand malformations. PAP is more common than PPD, whereas central polydactyly is very uncommon.

Lower Extremity Malformations.

Lower Extremity Malformations.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

KIF3B promotes a PI3K signaling gradient causing changes in a Shh protein gradient and suppressing polydactyly in mice.

Digit determination in limb buds is driven by a posteriorizing Sonic hedgehog (Shh) protein gradient; however, the mechanism regulating this is unclear. Here, we propose a diffusion-and-trapping hypothesis for Shh gradient formation based on data from the preaxial polydactyly phenotype of KIF3B motor hypomorphic mice. In the limb buds of these mice, a distal-to-proximal gradient of fibroblast growth factor (FGF) and phosphatidylinositol 3-kinase (PI3K) signaling and a posterior-to-anterior gradient of Shh were disorganized. This phenotype was reproduced by transplanting FGF8b-soaked beads. At the subcellular level, KIF3B transported the phosphatase and tensin homolog (PTEN)-like phosphatase Talpid3 to terminate PI3K signaling. High and low PI3K signaling strengths differentially sorted endocytosed Shh toward exosome-like particles and cytonemal punctata, respectively. These results indicate that the Shh-containing particles undergo either the diffusional movement in the periphery or cytonemal trapping in the center and form a spatial gradient along the periphery of developing limb buds.

ROR2-Vangl2 complexes direct ovarian cancer cell motility in response to Wnt5a (265)

<b>Objectives:</b> In both developmental and cancer contexts, collective migration has shown to be at least partially dependent upon asymmetric localization of core protein complexes within leader and follower cells. A critical function of non-canonical Wnt signaling in vertebrates is to regulate planar cell polarity (PCP), the polarity axis that organizes the plane of a tissue. Cells at the invasive front of cellular aggregates exhibit elevated expression of genes associated with motility pathways, including Wnt/PCP signaling. Using microfluidic devices, we have previously shown the soluble non-canonical Wnt ligand, <i>WNT5A</i>, influencing the directional motion of ovarian cancer cells via the ROR2 receptor. We sought to determine whether asymmetrical localization of ROR2 could guide <i>WNT5A-</i>directed motility. <b>Methods:</b> A DNA sequence encoding a fusion protein of ROR2 with a C-terminal Emerald (Em) fluorescent protein tag was introduced into ES2 ovarian cancer cells. Aggregated spheroids of ES2 ROR2-Em cells were plated onto a fibronectin-coated surface, and time-lapse fluorescent images were obtained of the radial migration in the presence or absence of <i>WNT5A</i>. Based on the direction of motion, cells were divided into leading or trailing half for analysis of the localization of ROR2 protein. Data were presented as mean +/- SEM. For co-immunoprecipitation experiments, ES2 cells expressing a Myctagged ROR2 DNA construct were utilized, with Myc epitope used for pulldown. <b>Results:</b> In the absence of <i>WNT5A</i>, ROR2 was uniformly distributed among the leading and lagging edge of the migrating cells (46.9% +/-2.452 ROR2 localized on the leading edge). In the presence of <i>WNT5A</i>, ROR2 was preferentially located to the leading edge of migrating cells (64.6% +/- 3.908 ROR2 localized on the leading edge). <i>VANGL2</i>, a core component in planar cell polarity, formed Wnt-induced receptor complexes with ROR2 in developing limb buds and other tumor types. <i>VANGL2</i> had been shown to reside at the protrusive edge of leader cells. To address whether <i>VANGL2</i>-ROR2 complexes contributed to the leading-edge localization of ROR2 in ovarian cancer cells, co-IP experiments were performed. Complex formation of ROR2-<i>VANGL2</i> was present in ovarian cancer cells at baseline (<i>VANGL2</i>:ROR2 IP ratio 1), yet was increased in the presence of <i>WNT5A</i> (<i>VANGL2</i>:ROR2 IP ratio 1.5). <b>Conclusions:</b> We have previously shown that <i>WNT5A</i> promotes the motility of ovarian cancer cells by influencing both directionality of motion as well as the degree of displacement in a ROR2 dependent manner. Asymmetric localization of Wnt/PCP core component complexes within leader and follower cells is crucial for cell motility, an intrinsic metastatic property. Our studies suggest that under the influence of <i>WNT5A</i>, ROR2 localizes to the leading edge of migrating cells. Additionally, ROR2 complexes with <i>VANGL2</i> in ovarian cancer cells, and this complex is enhanced in the presence of <i>WNT5A</i>. As <i>WNT5A</i> is produced at high levels by peritoneal mesothelial cells and omental adipose tissue in the microenvironment of ovarian cancer metastasis, disruption of the ROR2-<i>VANGL2</i> complex represents an attractive target for disrupting metastatic colonization of ovarian tumors.

Novel bilateral symmetrical congenital transverse upper and lower limb deficiencies in siblings in Ethiopia: two case reports

BackgroundTransverse congenital limb deficiency is a common limb deficiency where there is normal limb development until a certain point, beyond which no anatomical structure exists. Typically, this presents as an isolated and spontaneous abnormality as a result of arrest during limb bud development. Transverse bilateral deficiency in both upper and lower limbs is not well described.Case presentationWe report the cases of two female Ethiopian amhara siblings, aged 6 years and 5 months, respectively, from Ethiopia with similar transverse bilateral upper and lower limb deficiencies. The sisters were born from the same parents and have similar phenotypic presentations. Neither of them have other syndromic features or systemic manifestations. The siblings are currently on follow-up and are receiving assistance by specialist orthotists, who are working to improve walking and also providing adaptive equipment to facilitate self-care and feeding.ConclusionThe relationship of the patients and the similarity of phenotypical presentations suggests a strong genetic link.

Radial Polydactyly.

» Radial polydactyly is one of the most common congenital anomalies of the hand, with an incidence of 0.08 to 1.40 per 1,000 live births; it requires surgical treatment early in life.» Polydactyly occurs during weeks 5, 6, 7, and 8 of embryogenesis, principally due to misregulation of the sonic hedgehog protein within the developing limb bud.» The Flatt classification system of preaxial polydactyly (types I to VII), as published by Wassel, categorizes preaxial polydactyly based on osseous abnormalities, but it has substantial limitations.» For improved function and appearance, preaxial polydactyly ideally requires surgical intervention at an early age (between 1 and 2 years of age) before the development of fine motor skills.

Ontogenetic systematic characterisation of an endemic frog Rhacophorus malabaricus Jerdon, 1870 (Anura: Rhacophoridae) from Western Ghats, Kerala, India

In the present study, the complete description of the development and metamorphosis of an endemic frog Rhacophorus malabaricus, Jerdon, 1870, is documented in its natural habitat from the Southern part of Western Ghats (Peppara Wildlife Sanctuary, India). A brief illustration of each Gosner stage (fertilized egg to metamorphosed froglet) is given based on the direct monitoring and characterisation of morphological and morphometric variations. The identity of the tadpoles was confirmed by gene sequencing. Fertilisation and early development (cleavage, blastulation gastrulation and neurulation) take place inside the foam at 21.4 ± 0.1°C. The motile stage begins at 3rd day, after hatching and then eventually dropping into the water body. Hind limbs start differentiation first on the 15th day. The larva attains maximum size (TL 48.70 ± 0.22 mm) at Gosner stages 42. The morphometric measurements are significantly correlated with Gosner stages especially from 26 to 39. Oral disc features are described with LTRF of 7(3-7)/3 and KRF of 2:5+5/3. The morphological and morphometrical data of R. malabaricus larva is compared with other known Rhacophorus Kuhl & Van Hasselt, 1822 members. The present study shows that the relative lengths of TL, SVL, BH and TAL with stages are significant morphometric characters for taxonomy. Morphological features (limb bud development, pigmentation, oral disc features, etc.) are potentially useful characters for tadpole based anuran taxonomy at stage 26 or later.

Global DNA methylation and chondrogenesis of rat limb buds in a three-dimensional organ culture system.

Although DNA methylation epigenetically regulates development, data on global DNA methylation during the development of limb buds (LBs) are scarce. We aimed to investigate the global DNA methylation developmental dynamics in rat LBs cultivated in a serum supplemented (SS) and in chemically defined serum- and protein-free (SF) three-dimensional organ culture. Fischer rat front- and hind-LBs at the 13th and 14th gestation days (GDs) were cultivated at the air-liquid interface in Eagle’s Minimal Essential Medium (MEM) or MEM with 50% rat serum for 14 days, as SF and SS conditions, respectively. The methylation of repetitive DNA sequences (short interspersed DNA repetitive element [SINE] rat ID elements) was assessed by pyrosequencing. Development was evaluated by light microscopy and extracellular matrix glycosaminoglycans staining by safranin O. On isolation, weak safranin O staining was present only in more developed GD14 front-LBs. Chondrogenesis proceeded well in all cultures toward day 14, except in the SF-cultivated GD13 hind-LBs, where safranin O staining was almost absent on day 3 that was associated with a higher percentage of DNA methylation than in SF-cultivated GD13 front-LBs on day 3. In SF-cultivated front-LBs, a significant methylation increase between the 3rd and 14th days was detected. In SS-cultivated GD13 front-LBs, methylation increased significantly on day 3 and then decreased. In older GD14 SS-cultivated LBs, there was no increase of DNA methylation, but they were significantly hypomethylated relative to the SS-cultivated GD13 at days 3 and 14. We confirmed that the global DNA methylation increase is associated with less developed limb organ primordia that strive toward differentiation in vitro, which is of importance for regenerative medicine strategies.