The significance of ultradian oscillations in development.

Genes regulating developmental processes have been identified, but the mechanisms underlying their expression with the correct timing are still under investigation. Several genes show oscillatory expression that regulates the timing of developmental processes, such as somitogenesis and neurogenesis. These oscillations are also important for other developmental processes, such as cell proliferation and differentiation. In this review, we discuss the significance of oscillatory gene expression in developmental time and other forms of regulation.

Bioinformatics Analysis and Experimental Verification Identify Downregulation of COL27A1 in Poor Segmental Congenital Scoliosis.

BACKGROUND: Congenital scoliosis (CS) represents the congenital defect disease, and poor segmental congenital scoliosis (PSCS) represents one of its types. Delayed intervention can result in disability and paralysis. In this study, we would identify the core biomarkers for PSCS progression through bioinformatics analysis combined with experimental verification. METHODS: This work obtained the GSE11854 expression dataset associated with somite formation in the GEO database, which covers data of 13 samples. Thereafter, we utilized the edgeR of the R package to obtain DEGs in this dataset. Then, GO annotation, KEGG analyses, and DO annotation of DEGs were performed by "clusterProfiler" of the R package. This study performed LASSO regression for screening the optimal predicting factors for somite formation. Through RNA sequencing based on peripheral blood samples from healthy donors and PSCS cases, we obtained the RNA expression patterns and screen out DEGs using the R package DESeq2. The present work analyzed COL27A1 expression in PSCS patients by the RT-PCR assay. RESULTS: A total of 443 genes from the GSE11854 dataset were identified as DEGs, which were involved in BP associated with DNA replication, CC associated with chromosomal region, and MF associated with ATPase activity. These DEGs were primarily enriched in the TGF-ß signaling pathway and spinal deformity. Further, LASSO regression suggested that 9 DEGs acted as the signature markers for somite formation. We discovered altogether 162 DEGs in PSCS patients, which were involved in BP associated with cardiac myofibril assembly and MF associated with structural constituent of muscle. However, these 162 DEGs were not significantly correlated with any pathways. Finally, COL27A1 was identified as the only intersected gene between the best predictors for somite formation and PSCS-related DEGs, which was significantly downregulated in PSCS patients. CONCLUSION: This work sheds novel lights on DEGs related to the PSCS pathogenic mechanism, and COL27A1 is the possible therapeutic target for PSCS. Findings in this work may contribute to developing therapeutic strategies for PSCS.

A segmentation clock patterns cellular differentiation in a bacterial biofilm.

Contrary to multicellular organisms that display segmentation during development, communities of unicellular organisms are believed to be devoid of such sophisticated patterning. Unexpectedly, we find that the gene expression underlying the nitrogen stress response of a developing Bacillus subtilis biofilm becomes organized into a ring-like pattern. Mathematical modeling and genetic probing of the underlying circuit indicate that this patterning is generated by a clock and wavefront mechanism, similar to that driving vertebrate somitogenesis. We experimentally validated this hypothesis by showing that predicted nutrient conditions can even lead to multiple concentric rings, resembling segments. We additionally confirmed that this patterning mechanism is driven by cell-autonomous oscillations. Importantly, we show that the clock and wavefront process also spatially patterns sporulation within the biofilm. Together, these findings reveal a biofilm segmentation clock that organizes cellular differentiation in space and time, thereby challenging the paradigm that such patterning mechanisms are exclusive to plant and animal development.

Circadian key component CLOCK/BMAL1 interferes with segmentation clock in mouse embryonic organoids.

In mammals, circadian clocks are strictly suppressed during early embryonic stages, as well as in pluripotent stem cells, by the lack of CLOCK/BMAL1-mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, and with these, the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process, and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis implied that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.

What we can learn from embryos to understand the mesenchymal-to-epithelial transition in tumor progression.

Epithelial plasticity involved the terminal and transitional stages that occur during epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET), both are essential at different stages of early embryonic development that have been co-opted by cancer cells to undergo tumor metastasis. These processes are regulated at multiple instances, whereas the post-transcriptional regulation of key genes mediated by microRNAs is gaining major attention as a common and conserved pathway. In this review, we focus on discussing the latest findings of the cellular and molecular basis of the less characterized process of MET during embryonic development, with special attention to the role of microRNAs. Although we take in consideration the necessity of being cautious when extrapolating the obtained evidence, we propose some commonalities between early embryonic development and cancer progression that can shed light into our current understanding of this complex event and might aid in the design of specific therapeutic approaches.

Imaging and manipulating the segmentation clock.

During embryogenesis, the processes that control how cells differentiate and interact to form particular tissues and organs with precise timing and shape are of fundamental importance. One prominent example of such processes is vertebrate somitogenesis, which is governed by a molecular oscillator called the segmentation clock. The segmentation clock system is initiated in the presomitic mesoderm in which a set of genes and signaling pathways exhibit coordinated spatiotemporal dynamics to establish regularly spaced boundaries along the body axis; these boundaries provide a blueprint for the development of segment-like structures such as spines and skeletal muscles. The highly complex and dynamic nature of this in vivo event and the design principles and their regulation in both normal and abnormal embryogenesis are not fully understood. Recently, live-imaging has been used to quantitatively analyze the dynamics of selected components of the circuit, particularly in combination with well-designed experiments to perturb the system. Here, we review recent progress from studies using live imaging and manipulation, including attempts to recapitulate the segmentation clock in vitro. In combination with mathematical modeling, these techniques have become essential for disclosing novel aspects of the clock.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Insight into unique somitogenesis of yak (Bos grunniens) with one additional thoracic vertebra.

BACKGROUND: The yak is a species of livestock which is crucial for local communities of the Qinghai-Tibet Plateau and adjacent regions and naturally owns one more thoracic vertebra than cattle. Recently, a sub-population of yak termed as the Jinchuan yak has been identified with over half its members own a thoracolumbar vertebral formula of T15L5 instead of the natural T14L5 arrangement. The novel T15L5 positioning is a preferred genetic trait leading to enhanced meat and milk production. Selective breeding of this trait would have great agricultural value and exploration of the molecular mechanisms underlying this trait would both accelerate this process and provide us insight into the development and regulation of somitogenesis. RESULTS: Here we investigated the genetic background of the Jinchuan yak through resequencing fifteen individuals, comprising five T15L5 individuals and ten T14L5 individuals with an average sequencing depth of > 10X, whose thoracolumbar vertebral formulae were confirmed by anatomical observation. Principal component analysis, linkage disequilibrium analysis, phylogenetic analysis, and selective sweep analysis were carried out to explore Jinchuan yak's genetic background. Three hundred and thirty candidate markers were identified as associated with the additional thoracic vertebrae and target sequencing was used to validate seven carefully selected markers in an additional 51 Jinchuan yaks. The accuracies of predicting 15 thoracic vertebrae and 20 thoracolumbar vertebrae with these 7 markers were 100.00 and 33.33% despite they both could only represent 20% of all possible genetic diversity. Two genes, PPP2R2B and TBLR1, were found to harbour the most candidate markers associated with the trait and likely contribute to the unique somitic number and identity according to their reported roles in the mechanism of somitogenesis. CONCLUSIONS: Our findings provide a clear depiction of the Jinchuan yak's genetic background and a solid foundation for marker-assistant selection. Further exploitation of this unique population and trait could be promoted with the aid of our genomic resource.

Myostatin promotes the epithelial-to-mesenchymal transition of the dermomyotome during somitogenesis.

BACKGROUND: Myostatin (MSTN), a member of the transforming growth factor-ß (TGF-ß) superfamily, has been implicated in the negative regulation of skeletal myogenesis. However, the molecular mechanism through which MSTN regulates early embryonic myogenesis is not well understood. RESULTS: We demonstrate that MSTN regulates early embryonic myogenesis by promoting the epithelial-to-mesenchymal transition (EMT) of the dermomyotome during somitogenesis in chicks. We show that the MSTN gene is first expressed at the center of the dermomyotome. As somitogenesis progresses, its expression extends dorsally and ventrally along the plane of the dermomyotome. By combining in situ hybridization and immunofluorescence assays, we demonstrate that the expression pattern of MSTN is spatiotemporally well correlated with EMT of the dermomyotome. Our gain- and loss-of-function experiments further reveal that MSTN can induce EMT of the chick dermomyotome. We also show that MSTN induces EMT of a nonsmall cell lung carcinoma cell line (A549) and Madin-Darby canine kidney cells in vitro. CONCLUSIONS: Our experimental data suggest that MSTN regulates myogenesis by promoting EMT during somitogenesis. These findings provide novel insights into the functions of MSTN during early embryonic myogenesis. Developmental Dynamics 247:1241-1252, 2018. © 2018 Wiley Periodicals, Inc.

Functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior presomitic mesoderm in zebrafish.

Somites sequentially form with a regular interval by the segmentation from the anterior region of the presomitic mesoderm (PSM). The expression of several genes involved in the somite segmentation is switched off at the transition from the anterior PSM to somites. Zebrafish Ripply1, which down-regulates a T-box transcription factor Tbx6, is required for the suppression of segmentation gene expression. However, the functional roles of the Ripply-mediated suppression of segmentation gene expression at the anterior PSM remain elusive. In this study, we generated ripply1 mutants and examined genetic interaction between ripply1/2 and tbx6. Zebrafish ripply1-/- embryos failed to form the somite boundaries as was observed in knockdown embryos. We found that somite segmentation defects in ripply1 mutants were suppressed by heterozygous mutation of tbx6 or partial translational inhibition of tbx6 by antisense morpholino. We further showed that somite boundaries that were recovered in tbx6+/-; ripply1-/- embryos were dependent on the function of ripply2, indicating that relative gene dosage between ripply1/2 and tbx6 plays a critical role in the somite formation. Interestingly, the expression of segmentation genes such mesp as was still not fully suppressed at the anterior PSM of tbx6+/-; ripply1-/- embryos although the somite formation and rostral-caudal polarity of somites were properly established. Furthermore, impaired myogenesis was observed in the segmented somites in tbx6+/-; ripply1-/- embryos. These results revealed that partial suppression of the segmentation gene expression by Ripply is sufficient to establish the rostral-caudal polarity of somites, and that stronger suppression of the segmentation gene expression by Ripply is required for proper myogenesis in zebrafish embryos.

Spiral waves and vertebrate embryonic handedness.

During early embryonic development, the vertebrate main body axis is segmented from head-to-tail into somites. Somites emerge sequentially from the presomitic mesoderm (PSM) as a consequence of oscillatory waves of genetic activity, called somitogenesis waves. Here, we discuss the implications of the dynamic patterns of early X-Delta-2 expression in the prospective somites (somitomeres) of Xenopus laevis. We report that right somitomeres normally emerge before left to form chiral structures (i.e. structures having clockwise or counter-clockwise handedness). From our observations, we infer that somitogenesis waves are normally counter-clockwise spirals, a novel dynamic mechanism for the control of handedness development in Xenopus. We propose that the same mechanism could control handedness development in all vertebrate embryos, providing a dynamical basis for the current asymmetric molecular transport model for generating left-right asymmetry.

Characterizing early embryonic development of Brown Tsaiya Ducks (Anas platyrhynchos) in comparison with Taiwan Country Chicken (Gallus gallus domestics).

Avian embryos are among the most convenient and the primary representatives for the study of classical embryology. It is well-known that the hatching time of duck embryos is approximately one week longer than that of chicken embryos. However, the key features associated with the slower embryonic development in ducks have not been adequately described. This study aimed to characterize the pattern and the speed of early embryogenesis in Brown Tsaiya Ducks (BTD) compared with those in Taiwan Country Chicken (TCC) by using growth parameters including embryonic crown-tail length (ECTL), primitive streak formation, somitogenesis, and other development-related parameters, during the first 72 h of incubation. Three hundred and sixty eggs from BTD and TCC, respectively, were incubated at 37.2°C, and were then dissected hourly to evaluate their developmental stages. We found that morphological changes of TCC embryos shared a major similarity with that of the Hamburger and Hamilton staging system during early chick embryogenesis. The initial primitive streak in TCC emerged between 6 and 7 h post-incubation, but its emergence was delayed until 10 to 13 h post-incubation in BTD. Similarly, the limb primordia (wing and limb buds) were observed at 51 h post-incubation in TCC embryos compared to 64 h post-incubation in BTD embryos. The allantois first appeared around 65 to 68 h in TCC embryos, but it was not observed in BTD embryos. At the 72 h post-incubation, 40 somites were clearly formed in TCC embryos while only 32 somites in BTD embryos. Overall, the BTD embryos developed approximately 16 h slower than the chicken embryo during the first 72 h of development. To our best knowledge, this is the first study to describe two distinct developmental time courses between TCC and BTD, which would facilitate future embryogenesis-related studies of the two important avian species in Taiwan.

TiO2 nanoparticles induce omphalocele in chicken embryo by disrupting Wnt signaling pathway.

Titanium dioxide nanoparticles (TiO2 NPs) are among abundantly used metal oxide NPs but their interactions with biomolecules and subsequent embryonic toxicity in higher vertebrates is not extensively reported. Physicochemical interactions of TiO2 NPs with egg albumen reveals that lower doses of TiO2 NPs (10 and 25 µg/ml) accounted for higher friccohesity and activation energy but an increment in molecular radii was recorded at higher doses (50 and 100 µg/ml). FTIR analysis revealed conformational changes in secondary structure of egg albumen as a result of electrostratic interactions between egg albumen and TiO2 NPs. The morphometric data of chicken embryo recorded a reduction at all the doses of TiO2 NPs, but toxicity and developmental deformity (omphalocele and flexed limbs) were recorded at lower doses only. Inductively coupled plasma optical emission spectrometry (ICP-OES) confirmed presence of Ti in chicken embryos. mRNA levels of genes involved in canonical and non-canonical Wnt signaling were lowered following TiO2 NPs treatment resulting in free radical mediated disruption of lateral plate mesoderm and somite myogenesis. Conformational changes in egg albumen and subsequent developmental deformity in chicken embryo following TiO2 NPs treatment warrants detailed studies of NP toxicity at lower doses prior to their biomedical applications.

Tail reduction process during human embryonic development.

Although the human tail is completely absent at birth, the embryonic tail is formed just as in other tailed amniotes. Since all morphological variations are created from variations in developmental processes, elucidation of the tail reduction process during embryonic development may be necessary to clarify the human evolutionary process. The tail has also been of great interest to the medical community. The congenital anomaly referred to as 'human tail', i.e. the occurrence of a tail-like structure, has been reported and was thought to represent a vestige of the embryonic tail; however, this hypothesis has not been verified. Accordingly, in this study, we aimed to establish a new method to visualize all somites in an embryo. We used sagittal-sectioned embryos from Carnegie Stage (CS) 13 to CS23. All samples were obtained from the Congenital Anomaly Research Center, Kyoto University, Japan. Combining photomicroscopy and three-dimensional reconstruction, we clearly visualized and labeled all somites. We found that the number of somites peaked at CS16 and dramatically decreased by approximately five somites. Tail reduction with a decrease in somites has also been observed in other short-tailed amniotes; thus, this result suggested the possibility that there is a common mechanism for morphogenesis of short tails in amniote species. Additionally, our findings provided important insights into the cause of the congenital anomaly known as 'human tail'.

Pofut1 point-mutations that disrupt O-fucosyltransferase activity destabilize the protein and abolish Notch1 signaling during mouse somitogenesis.

The segmental pattern of the vertebrate body is established via the periodic formation of somites from the presomitic mesoderm (PSM). This periodical process is controlled by the cyclic and synchronized activation of Notch signaling in the PSM. Protein O-fucosyltransferase1 (Pofut1), which transfers O-fucose to the EGF domains of the Notch1 receptor, is indispensable for Notch signaling activation. The Drosophila homologue Ofut1 was reported to control Notch localization via two different mechanisms, working as a chaperone for Notch or as a regulator of Notch endocytosis. However, these were found to be independent of O-fucosyltransferase activity because the phenotypes were rescued by Ofut1 mutants lacking O-fucosyltransferase activity. Pofut1 may also be involved in the Notch receptor localization in mice. However, the contribution of enzymatic activity of Pofut1 to the Notch receptor dynamics remains to be elucidated. In order to clarify the importance of the O-fucosyltransferase activity of Pofut1 for Notch signaling activation and the protein localization in the PSM, we established mice carrying point mutations at the 245th a.a. or 370-372th a.a., highly conserved amino-acid sequences whose mutations disrupt the O-fucosyltransferase activity of both Drosophila Ofut1 and mammalian Pofut1, with the CRISPR/Cas9 mediated genome-engineering technique. Both mutants displayed the same severely perturbed somite formation and Notch1 subcellular localization defects as the Pofut1 null mutants. In the mutants, Pofut1 protein, but not RNA, became undetectable by E9.5. Furthermore, both wild-type and mutant Pofut1 proteins were degraded through lysosome dependent machinery. Pofut1 protein loss in the point mutant embryos caused the same phenotypes as those observed in Pofut1 null embryos.

The WHHERE coactivator complex is required for retinoic acid-dependent regulation of embryonic symmetry.

Bilateral symmetry is a striking feature of the vertebrate body plan organization. Vertebral precursors, called somites, provide one of the best illustrations of embryonic symmetry. Maintenance of somitogenesis symmetry requires retinoic acid (RA) and its coactivator Rere/Atrophin2. Here, using a proteomic approach we identify a protein complex, containing Wdr5, Hdac1, Hdac2 and Rere (named WHHERE), which regulates RA signaling and controls embryonic symmetry. We demonstrate that Wdr5, Hdac1, and Hdac2 are required for RA signaling in vitro and in vivo. Mouse mutants for Wdr5 and Hdac1 exhibit asymmetrical somite formation characteristic of RA-deficiency. We also identify the Rere-binding histone methyltransferase Ehmt2/G9a, as a RA coactivator controlling somite symmetry. Upon RA treatment, WHHERE and Ehmt2 become enriched at RA target genes to promote RNA polymerase II recruitment. Our work identifies a protein complex linking key epigenetic regulators acting in the molecular control of embryonic bilateral symmetry.Retinoic acid (RA) regulates the maintenance of somitogenesis symmetry. Here, the authors use a proteomic approach to identify a protein complex of Wdr5, Hdac1, Hdac2 that act together with RA and coactivator Rere/Atrophin2 and a histone methyltransferase Ehmt2 to regulate embryonic symmetry.

DYRK2 displays muscle fiber type specific function during zebrafish early somitogenesis.

Dual specificity tyrosine-phosphorylation regulated kinase 2 (DYRK2) is a serine/threonine kinase. In zebrafish, DYRK2 is expressed in the lateral somites and adaxial cells at the early stage of embryo development. However, its role in early myogenesis had not been elucidated yet. Here, we report that DYRK2 mRNA and MyoD mRNA were colocalized in the muscle progenitor cells in somites, including both the posterior compartment of the lateral somites and adaxial cells. Knockdown of DYRK2 reduced the levels of MyoD transcripts in the muscle progenitor cells in somites. In contrast, overexpression of DYRK2 increased the levels of MyoD transcripts in the muscle progenitor cells in somites. The effects of knockdown and overexpression of DYRK2 on the expression of MyoD in the posterior compartment of the lateral somites were much greater than in the adaxial cells. Further studies indicated that forced expression of DYRK2 increased the levels of fast-twitch skeletal myosin RNA. Moreover, knockdown or forced expression of DYRK2 affected the levels of fast-twitch skeletal myosin protein. Together, these data indicate that DYRK2 is expressed in the developing muscle progenitor cells in somites and that it positively regulates fast-twitch muscle differentiation, at least at the early stages.

Knockdown of epigenetic transcriptional co-regulator Brd2a disrupts apoptosis and proper formation of hindbrain and midbrain-hindbrain boundary (MHB) region in zebrafish.

Brd2 is a member of the bromodomain-extraterminal domain (BET) family of proteins and functions as an acetyl-histone-directed transcriptional co-regulator and recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. While Brd2 acts as a protooncogene in mammalian blood, developmental studies link it to regulation of neuronal apoptosis and epilepsy, and complete knockout of the gene is invariably embryonic lethal. In Drosophila, the Brd2 homolog acts as a maternal effect factor necessary for segment formation and identity and proper expression of homeotic loci, including Ultrabithorax and engrailed. To test the various roles attributed to Brd2 in a single developmental system representing a non-mammalian vertebrate, we conducted a phenotypic characterization of Brd2a deficient zebrafish embryos produced by morpholino knockdown and corroborated by Crispr-Cas9 disruption and small molecule inhibitor treatments. brd2aMO morphants exhibit reduced hindbrain with an ill-defined midbrain-hindbrain boundary (MHB) region; irregular notochord, neural tube, and somites; and abnormalities in ventral trunk and ventral nerve cord interneuron positioning. Using whole mount TUNEL and confocal microscopy, we uncover a significant decrease, then a dramatic increase, of p53-independent cell death at the start and end of segmentation, respectively. In contrast, using qualitative and quantitative analyses of BrdU incorporation, phosphohistone H3-tagging, and flow cytometry, we detect little effect of Brd2a knockdown on overall proliferation levels in embryos. RNA in situ hybridization shows reduced or absent expression of homeobox gene eng2a and paired box gene pax2a, in the hindbrain domain of the MHB region, and an overabundance of pax2a-positive kidney progenitors, in knockdowns. Together, these results suggest an evolutionarily conserved role for Brd2 in the proper formation and/or patterning of segmented tissues, including the vertebrate CNS, where it acts as a bi-modal regulator of apoptosis, and is necessary, directly or indirectly, for proper expression of genes that pattern the MHB and/or regulate differentiation in the anterior hindbrain.

Tbx5 Buffers Inherent Left/Right Asymmetry Ensuring Symmetric Forelimb Formation.

The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in TBX5, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor Tbx5 in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of Tbx5 produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In Tbx hypomorphic mutants maintained on an INV mutant background, with situs inversus, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of Tbx5 are required to overcome this asymmetry to ensure symmetric forelimb formation.