Developing Mouse Limb Bud Research Articles

A p53-dependent translational program directs tissue-selective phenotypes in a model of ribosomopathies.

In ribosomopathies, perturbed expression of ribosome components leads to tissue-specific phenotypes. What accounts for such tissue-selective manifestations as a result of mutations in the ribosome, a ubiquitous cellular machine, has remained a mystery. Combining mouse genetics and invivo ribosome profiling, we observe limb-patterning phenotypes in ribosomal protein (RP) haploinsufficient embryos, and we uncover selective translational changes of transcripts that controlling limb development. Surprisingly, both loss of p53, which is activated by RP haploinsufficiency, and augmented protein synthesis rescue these phenotypes. These findings are explained by the finding that p53 functions as a master regulator of protein synthesis, at least in part, through transcriptional activation of 4E-BP1. 4E-BP1, a key translational regulator, in turn, facilitates selective changes in the translatome downstream of p53, and this thereby explains how RP haploinsufficiency may elicit specificity to gene expression. These results provide an integrative model to help understand how invivo tissue-specific phenotypes emerge in ribosomopathies.

Ptch2 shares overlapping functions with Ptch1 in Smo regulation and limb development

Ptch1 and Ptch2 are highly conserved vertebrate homologs of Drosophila ptc, the receptor of the Hedgehog (Hh) signaling pathway. The vertebrate Ptch1 gene encodes a potent tumor suppressor and is well established for its role in embryonic development. In contrast, Ptch2 is poorly characterized and dispensable for embryogenesis. In flies and mice, ptc/Ptch1 controls Hh signaling through the regulation of Smoothened (Smo). In addition, Hh pathway activation also up-regulates ptc/Ptch1 expression to restrict the diffusion of the ligand. Recent studies have implicated Ptch2 in this ligand dependent antagonism, however whether Ptch2 encodes a functional Shh receptor remains unclear. In this report, we demonstrate that Ptch2 is a functional Shh receptor, which regulates Smo localization and activity in vitro. We also show that Ptch1 and Ptch2 are co-expressed in the developing mouse limb bud and loss of Ptch2 exacerbates the outgrowth defect in the limb-specific Ptch1 knockout mutants, demonstrating that Ptch1 and Ptch2 co-operate in regulating cellular responses to Shh in vivo.

Tbx2 Terminates Shh/Fgf Signaling in the Developing Mouse Limb Bud by Direct Repression of Gremlin1

Vertebrate limb outgrowth is driven by a positive feedback loop that involves Sonic hedgehog (Shh) and Gremlin1 (Grem1) in the posterior limb bud mesenchyme and Fibroblast growth factors (Fgfs) in the overlying epithelium. Proper spatio-temporal control of these signaling activities is required to avoid limb malformations such as polydactyly. Here we show that, in Tbx2-deficient hindlimbs, Shh/Fgf4 signaling is prolonged, resulting in increased limb bud size and duplication of digit 4. In turn, limb-specific Tbx2 overexpression leads to premature termination of this signaling loop with smaller limbs and reduced digit number as phenotypic manifestation. We show that Tbx2 directly represses Grem1 in distal regions of the posterior limb mesenchyme allowing Bone morphogenetic protein (Bmp) signaling to abrogate Fgf4/9/17 expression in the overlying epithelium. Since Tbx2 itself is a target of Bmp signaling, our data identify a growth-inhibiting positive feedback loop (Bmp/Tbx2/Grem1). We propose that proliferative expansion of Tbx2-expressing cells mediates self-termination of limb bud outgrowth due to their refractoriness to Grem1 induction.

The molecular basis of human congenital limb malformations

This review focuses predominantly on the human congenital malformations caused by alterations affecting the morphoregulatory gene networks that control early limb bud patterning and outgrowth. Limb defects are among the most frequent congenital malformations in humans that are caused by genetic mutations or teratogenic effects resulting either in abnormal, loss of, or additional skeletal elements. Spontaneous and engineered mouse models have been used to identify and study the molecular alterations and disrupted gene networks that underlie human congenital limb malformations. More recently, mouse genetics has begun to reveal the alterations that affect the often-large cis-regulatory landscapes that control gene expression in limb buds and cause devastating effects on limb bud development. These findings have paved the way to identifying mutations in cis-regulatory regions as causal to an increasing number of congenital limb malformations in humans. In these cases, no mutations in the coding region of a presumed candidate were previously detected. This review highlights how the current understanding of the molecular gene networks and interactions that control mouse limb bud development provides insight into the etiology of human congenital limb malformations.

Conserved cis-regulatory regions in a large genomic landscape control SHH and BMP-regulated Gremlin1expression in mouse limb buds

BackgroundMouse limb bud is a prime model to study the regulatory interactions that control vertebrate organogenesis. Major aspects of limb bud development are controlled by feedback loops that define a self-regulatory signalling system. The SHH/GREM1/AER-FGF feedback loop forms the core of this signalling system that operates between the posterior mesenchymal organiser and the ectodermal signalling centre. The BMP antagonist Gremlin1 (GREM1) is a critical node in this system, whose dynamic expression is controlled by BMP, SHH, and FGF signalling and key to normal progression of limb bud development. Previous analysis identified a distant cis-regulatory landscape within the neighbouring Formin1 (Fmn1) locus that is required for Grem1 expression, reminiscent of the genomic landscapes controlling HoxD and Shh expression in limb buds.ResultsThree highly conserved regions (HMCO1-3) were identified within the previously defined critical genomic region and tested for their ability to regulate Grem1 expression in mouse limb buds. Using a combination of BAC and conventional transgenic approaches, a 9 kb region located ~70 kb downstream of the Grem1 transcription unit was identified. This region, termed Grem1 Regulatory Sequence 1 (GRS1), is able to recapitulate major aspects of Grem1 expression, as it drives expression of a LacZ reporter into the posterior and, to a lesser extent, in the distal-anterior mesenchyme. Crossing the GRS1 transgene into embryos with alterations in the SHH and BMP pathways established that GRS1 depends on SHH and is modulated by BMP signalling, i.e. integrates inputs from these pathways. Chromatin immunoprecipitation revealed interaction of endogenous GLI3 proteins with the core cis-regulatory elements in the GRS1 region. As GLI3 is a mediator of SHH signal transduction, these results indicated that SHH directly controls Grem1 expression through the GRS1 region. Finally, all cis-regulatory regions within the Grem1 genomic landscape locate to the DNAse I hypersensitive sites identified in this genomic region by the ENCODE consortium.ConclusionsThis study establishes that distant cis-regulatory regions scattered through a larger genomic landscape control the highly dynamic expression of Grem1, which is key to normal progression of mouse limb bud development.

A Computational Clonal Analysis of the Developing Mouse Limb Bud

A comprehensive spatio-temporal description of the tissue movements underlying organogenesis would be an extremely useful resource to developmental biology. Clonal analysis and fate mappings are popular experiments to study tissue movement during morphogenesis. Such experiments allow cell populations to be labeled at an early stage of development and to follow their spatial evolution over time. However, disentangling the cumulative effects of the multiple events responsible for the expansion of the labeled cell population is not always straightforward. To overcome this problem, we develop a novel computational method that combines accurate quantification of 2D limb bud morphologies and growth modeling to analyze mouse clonal data of early limb development. Firstly, we explore various tissue movements that match experimental limb bud shape changes. Secondly, by comparing computational clones with newly generated mouse clonal data we are able to choose and characterize the tissue movement map that better matches experimental data. Our computational analysis produces for the first time a two dimensional model of limb growth based on experimental data that can be used to better characterize limb tissue movement in space and time. The model shows that the distribution and shapes of clones can be described as a combination of anisotropic growth with isotropic cell mixing, without the need for lineage compartmentalization along the AP and PD axis. Lastly, we show that this comprehensive description can be used to reassess spatio-temporal gene regulations taking tissue movement into account and to investigate PD patterning hypothesis.

Distinct Roles of Hand2 in Initiating Polarity and Posterior Shh Expression during the Onset of Mouse Limb Bud Development

The polarization of nascent embryonic fields and the endowment of cells with organizer properties are key to initiation of vertebrate organogenesis. One such event is antero-posterior (AP) polarization of early limb buds and activation of morphogenetic Sonic Hedgehog (SHH) signaling in the posterior mesenchyme, which in turn promotes outgrowth and specifies the pentadactylous autopod. Inactivation of the Hand2 transcriptional regulator from the onset of mouse forelimb bud development disrupts establishment of posterior identity and Shh expression, which results in a skeletal phenotype identical to Shh deficient limb buds. In wild-type limb buds, Hand2 is part of the protein complexes containing Hoxd13, another essential regulator of Shh activation in limb buds. Chromatin immunoprecipitation shows that Hand2-containing chromatin complexes are bound to the far upstream cis-regulatory region (ZRS), which is specifically required for Shh expression in the limb bud. Cell-biochemical studies indicate that Hand2 and Hoxd13 can efficiently transactivate gene expression via the ZRS, while the Gli3 repressor isoform interferes with this positive transcriptional regulation. Indeed, analysis of mouse forelimb buds lacking both Hand2 and Gli3 reveals the complete absence of antero-posterior (AP) polarity along the entire proximo-distal axis and extreme digit polydactyly without AP identities. Our study uncovers essential components of the transcriptional machinery and key interactions that set-up limb bud asymmetry upstream of establishing the SHH signaling limb bud organizer.

Fgf-Dependent Etv4/5 Activity Is Required for Posterior Restriction of Sonic hedgehog and Promoting Outgrowth of the Vertebrate Limb

Crosstalk between the fibroblast growth factor (FGF) and Sonic Hedgehog (Shh) pathways is critical for proper patterning and growth of the developing limb bud. Here, we show that FGF-dependent activation of the ETS transcription factors Etv4 and Etv5 contributes to proximal-distal limb outgrowth. Surprisingly, blockage of Etv activity in early distal mesenchyme also resulted in ectopic, anterior expansion of Shh, leading to a polydactylous phenotype. These data indicate an unexpected function for an FGF/Etv pathway in anterior-posterior patterning. FGF activity in the limb is not only responsible for maintaining posterior-specific Shh expression, but it also acts via Etvs to prevent inappropriate anterior expansion of Shh. This study identifies another level of genetic interaction between the orthogonal axes during limb development.

Identification of genes controlled by LMX1B in the developing mouse limb bud

In the developing limb, dorsal-ventral patterning is controlled by the transcription factor LMX1B, expressed in the dorsal mesenchyme. Loss of Lmx1b function in mice or humans results in the loss of dorsal limb structures and Nail-Patella syndrome, but the effectors through which LMX1B controls limb patterning are virtually unknown. Using microarrays to analyze the differential expression of mRNAs in wild-type vs. Lmx1b(-/-) limb buds, we have identified hundreds of genes as putative LMX1B targets. Analysis of a subset of these candidates by in situ mRNA localization has identified eight genes previously unknown to require Lmx1b for their dorsal-ventral restriction of expression in the limb. Furthermore, our results suggest that LMX1B controls different targets along the proximal-distal axis of the limb, and suggest the existence of a dorsal proximal limb region that is rich in mRNAs requiring Lmx1b for their expression.

A series of ENU-induced single-base substitutions in a long-range cis-element altering Sonic hedgehog expression in the developing mouse limb bud

Mammal–fish-conserved-sequence 1 (MFCS1) is a highly conserved sequence that acts as a limb-specific cis-acting regulator of Sonic hedgehog ( Shh) expression, residing 1 Mb away from the Shh coding sequence in mouse. Using gene-driven screening of an ENU-mutagenized mouse archive, we obtained mice with three new point mutations in MFCS1: M101116, M101117, and M101192. Phenotype analysis revealed that M101116 mice exhibit preaxial polydactyly and ectopic Shh expression at the anterior margin of the limb buds like a previously identified mutant, M100081. In contrast, M101117 and M101192 show no marked abnormalities in limb morphology. Furthermore, transgenic analysis revealed that the M101116 and M100081 sequences drive ectopic reporter gene expression at the anterior margin of the limb bud, in addition to the normal posterior expression. Such ectopic expression was not observed in the embryos carrying a reporter transgene driven by M101117. These results suggest that M101116 and M100081 affect the negative regulatory activity of MFCS1, which suppresses anterior Shh expression in developing limb buds. Thus, this study shows that gene-driven screening for ENU-induced mutations is an effective approach for exploring the function of conserved, noncoding sequences and potential cis-regulatory elements.

Fate map of mouse ventral limb ectoderm and the apical ectodermal ridge

The apical ectodermal ridge (AER) is a critical signaling center at the tip of the limb that promotes outgrowth. In mouse, formation of the AER involves a gradual restriction of AER gene expression from a broad ventral preAER domain to the tip of the limb, as well as progressive thickening of cells to form a multilayered epithelium. The AER is visible from embryonic day 10.5 to 13.5 (E10.5–E13.5) in the mouse forelimb. Previous short-term fate mapping studies indicated that, once a cell is incorporated into the AER, its descendents remain within the AER. In addition, some preAER cells appear to become incorporated into the ventral ectoderm. In the present study, we used an inducible CreER/loxP fate mapping approach in mouse to examine the long-term contribution of preAER cells to limb ventral ectoderm, as well as the ultimate fate of the mature AER cells. We used a CreER transgene that contains Msx2 regulatory sequences specific to the developing AER, and demonstrate by marking preAER cells that, at stage 2 of mouse limb bud development, the majority of the ventral ectoderm that protrudes from the body wall later covers only the paw. Furthermore, when Msx2-CreER-expressing preAER cells are marked after the onset of preAER gene expression, a similar domain of paw ventral ectoderm is marked at E16.5, in addition to the AER. Strikingly, mapping the long-term fate of cells that form the mature AER showed that, although this structure is indeed a distinct compartment, AER-derived cells are gradually lost after E12.5 and no cells remain by birth. A distinct dorsal/ventral boarder nevertheless is maintained in the ectoderm of the paw, with the distal-most border being located at the edge of the nail bed. These studies have uncovered new aspects of the cellular mechanisms involved in AER formation and in partitioning the ventral ectoderm in mouse limb.

Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer.

We have used a Prx1 limb enhancer to drive expression of Cre Recombinase in transgenic mice. This regulatory element leads to Cre expression throughout the early limb bud mesenchyme and in a subset of craniofacial mesenchyme. Crossing a murine line carrying this transgene to a reporter mouse harboring a floxed Cre-reporter cassette revealed that recombinase activity is first observed in the earliest limb bud at 9.5 dpc. By early to mid bud stages at 10.5 dpc recombination is essentially complete in all mesenchymal cells in the limb. Expression of the Cre recombinase was never detected in the limb bud ectoderm. The use of Prx1-Cre mice should facilitate analysis of gene function in the developing limb.

Cloning and Characterization of Two Cytoplasmic Dynein Intermediate Chain Genes in Mouse and Human

Cytoplasmic dynein is a large multisubunit microtubule-based motor protein, which mediates movement of numerous intracellular organelles. We report here the identification of the human homologue of cytoplasmic dynein intermediate chain 1 gene (DNCI1) located on human chromosome 7q21.3–q22.1. The mouse orthologue (Dnci1) was identified along with another highly related gene,Dnci2,and their RNAin situexpression patterns were examined during mouse embryogenesis.Dnci1was found to have a highly restricted expression domain in the developing forebrain as well as the peripheral nervous system (PNS), whileDnci2displayed a broad expression profile throughout the entire central nervous system and most of the PNS. A dynamic expression profile was also found forDnci2in the developing mouse limb bud. The data presented here provide a framework for the further analysis of the functional role ofDnci1andDnci2in mouse andDNCI1in human.

Basal lamina molecules are concentrated in myogenic regions of the mouse limb bud.

Molecular components of basal lamina, such as laminin, stimulate the differentiation of skeletal muscle cells in culture, while interstitial matrix components such as fibronectin are inhibitory. However, the role of extracellular matrix (ECM) molecules in muscle cell differentiation in the embryo is less well understood. As a first step toward understanding the role of the ECM in embryonic myogenesis, the localization of basal lamina molecules in the mouse limb bud before and during muscle cell differentiation was determined by immunofluorescence. Laminin, collagen type IV and nidogen (entactin) were concentrated in myogenic regions of the limb bud both before and during differentiation of skeletal muscle cells. Punctate immunofluorescence for basal lamina molecules was concentrated in dorsal and ventral premuscle and muscle masses, when compared with other regions of limb mesenchyme. In contrast, immunofluorescence for fibronectin, an interstitial extracellular matrix molecule, was decreased in premuscle and muscle masses. These results suggest that basal lamina components play an important stimulatory role in early stages of skeletal muscle differentiation in the developing mouse limb bud.

MRNA expression patterns of the IGF system during mouse limb bud development, determined by whole mount in situ hybridization

During limb development the primary limb bud requires various signals to differentiate. Insulin-like growth factor (IGF)-I and IGF-II serve as ubiquitous cellular growth promoters and are modulated by their binding proteins (IGFBPs), which inhibit or augment IGF bioavailability. This is the first study to give a complete overview of the mRNA expression patterns of Igf-1, Igf-2, type 1 Igf receptor ( Igf1r) and six Igf binding proteins (IGFBP-1–6) in embryonic mouse limbs, at various stages of development, by whole mount in situ hybridization (ISH). Our results show that all the members of the Igf system, except Igfbp-1 and -6, have specific spatio-temporal mRNA expression patterns. IGFBP-2 and -5 are found in the apical ectodermal ridge (AER), and IGF-I and IGFBP-4 in the region of the zone of polarizing activity (ZPA). IGF-II and IGF1R are found in regions of pre-cartilage formation. At 13.5 days post coitus (dpc) the IGF system colocalizes with apoptosis areas; IGFBP-2, -4 and -5 are found in the interdigital zone, while IGFBP-3 and IGF-I border this region. Furthermore, IGFBP-3, -4 and -5 are found in the phalangeal joint areas, at an early stage of joint formation. This supports the hypothesis that the IGF system may be involved in chondrogenic differentiation of mesenchyme and the regulation of apoptosis in the developing limb.

Expression profile of Gli family members and Shh in normal and mutant mouse limb development.

Gli genes represent a small family, encoding zinc-finger proteins of the Krüppel-type. The family consists of Gli(1), Gli2, and Gli3, all of which are expressed in the developing mouse limb bud. To assess the role of the Gli family and Sonic hedgehog (Shh) in mouse limb development, we compared the expression domains of all three Gli genes and of Shh. Although each Gli gene has its own distinct expression pattern in limb buds, at 10.5-11.5 dpc all three genes were found not to be expressed in the posterior region, the presumptive Shh expression domain. This transient mutually exclusive expression suggested a potential interaction between Gli genes and Shh. To address this matter, we analysed the expression of Gli genes and Shh in two polydactyly mouse mutants, Extra toes (Xt) and Hemimelic-extra toes (Hx) which express Shh ectopically in the anterior region of the limb field. Since Xt mice lack Gli3 expression, the ectopic Shh expression is genetically linked to the absence of Gli3. In Hx mice we found a down-regulation of Gli3 in the anterior region of the limb bud. In both mutants Gli2 expression pattern was not altered, whereas Gli1 expression was anteriorly up-regulated adjacent to the ectopic Shh domain. These results strongly suggest a positive regulation of Gli1 by Shh and a negative interaction between Shh and Gli3.

Distribution of nestin in the developing mouse limb bud in vivo and in micro-mass cultures of cells isolated from limb buds

Early skeletal muscle development is accompanied by changes in the composition of the cytoskeleton. In this report we analyze the distribution of the intermediate filament nestin in the developing mouse limb buds in vivo and in mesenchymal cells isolated from limb buds in vitro. The subcellular distribution of nestin mRNA and protein in muscle cells was also analyzed. We find a shift in nestin expression during early limb bud development. At embryonic day 11 (E11), low levels of nestin (protein) were expressed in the mesenchymal cells of the developing limb bud. Later, nestin mRNA and protein were down-regulated in the mesenchymal condensations undergoing chondrogenesis (E12 and E13), but remained expressed predominantly in the ectodermal cells and in the differentiating myoblasts. At E18, only muscle fibres, endothelial cells and nerves were nestin positive. This shift in expression was reproduced in vitro, in micro-mass cultures of mesenchymal cells. In E11 cultures, nestin protein was initially expressed in all cells. Upon formation of cartilage foci (after 2–3 days in culture), nestin immunoreactivity was not observed in cartilage, and low levels were detected in the cells located between the foci. A subpopulation of mono- and multinucleated cells, peripheral to the cartilage nodules, expressed the muscle-specific intermediate filament desmin protein together with high levels of nestin protein. The proportion of nestin-expressing cells could be changed by addition of specific signalling molecules. Insulin-like growth factors I and II (IGF I and II) increased the number of nestin-positive cells, while basic fibroblast growth factor (FGF) reduced the number of nestin-expressing cells. Finally, we present evidence for a different subcellular localization of nestin protein and mRNA: the mRNA is predominantly located in the ends of the muscle cell, whereas the protein is found in the central region. Intracellular localization of nestin mRNA may constitute an additional level of regulation of the cytoskeleton during muscle development.

Focal expression of glial cell line-derived neurotrophic factor in developing mouse limb bud.

Glial cell line-derived neurotrophic factor (GDNF) is known to support the survival of motoneurons in vitro and in vivo, as well as subpopulations of sensory neurons in vitro. To clarify the mechanisms by which GDNF supports these neurons, we examined the patterns of GDNF mRNA expression in relation to motor and sensory axons during early stages of mouse development. Between embryonic days (E) 10 and 12, a time when motor and sensory axons are entering the periphery, GDNF mRNA is expressed at high levels in a restricted region in proximal limb buds where axons converge and enter the limb. At later ages (E14-16), GDNF mRNA was detected in non-neuronal cells along peripheral nerve, in dermis, and in some muscles. To characterize cells that express GDNF in the proximal limb, GDNF expression in the forelimb was compared to expression patterns of two markers of muscle, Pax 3 and myogenin, as well as with the pan neurotrophin receptor (p75) which is expressed by Schwann cell precursors. We show that expression of GDNF in the proximal limb bud at E11-12 does not correlate with markers of muscle or Schwann cell precursors, which supports the idea that GDNF is expressed by mesenchymal cells in this region. Our results suggest that GDNF expression in proximal limb buds may function as a transient survival factor, particularly for motor neurons, before they reach their final targets. GDNF expression in muscle and dermis at later stages suggests that GDNF may have additional functions as motor and sensory neurons mature.

A duplicated zone of polarizing activity in polydactylous mouse mutants.

The positional signaling along the anteroposterior axis of the developing vertebrate limb is provided by the zone of polarizing activity (ZPA) located at the posterior margin. Recently, it was established that the Sonic hedgehog (Shh) mediates ZPA activity. Here we report that a new mouse mutant, Recombination induced mutant 4 (Rim4), and two old mutants, Hemimelic extra toes (Hx) and Extra toes (Xt), exhibit mirror-image duplications of the skeletal pattern of the digits. In situ hybridization of the embryos of these mutants revealed ectopic expression of Shh and fibroblast growth factor-4 (Fgf-4) genes at the anterior margin of limb buds. The new mutation, Rim4, was mapped to chromosome 6 with linkage to HoxAbut segregated from HoxA. No linkage to other known polydactylous mutations was detected. In this mutant, ectopic expression of the Hoxd-11 gene, thought to be downstream of ZPA, was also observed at the anterior margin of the limb buds. All results indicate the presence of an additional ZPA at the anterior margin of limb buds in these mutants. Thus, it appears that multiple endogenous genes regulate the spatial localization of the ZPA in the developing mouse limb bud.

Effects of Acetazolamide on the Development of Mouse Limb Buds In Vitro

AbstractForelimb buds of mouse embryos were cultured in a serum‐free BGJb medium by a roller system. At the beginning of culture, acetazolamide was added to the final concentration of 10−5 M or 10−4 M. After 6 days of culture, no morphological abnormalities of the developing forelimb buds could be detected. In morphometrical analyses, the morphological scores of antebrachium and hand in the right forelimb of female embryos were 1.8 and 2.2 in the 10−5 M and 10−4 M acetazolamide‐treated groups, respectively, and 1.6 in controls. There was no difference in the size of the forelimb among these groups. Concerning cartilage skeleton, the incidence of bending of the ulnoradius was increased to 64–90% in 10−4 M acetazolamide‐treated group, which was different from the control (0.22%). In addition, there was a significant difference in the protein and DNA contents in only right forelimb buds between the acetazolamide‐treated and control groups. However, there were no differences in sex or laterality in the development of forelimbs and cartilage skeleton. No pathological changes in the cartilage skeleton and mesenchyme were observed in the acetazolamide‐treated forelimbs. From these findings, there is the possibility that acetazolamide per se may primarily interfere with the development of the forelimb in mouse embryos at the molecular level in vitro.