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.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP-associated variants in embryonic zebrafish.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins. RESULTS: Here, we performed in vivo functional characterization of human ACVR1R206H and orthologous zebrafish Acvr1lR203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1R206H and zebrafish Acvr1lR203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1R206H signaling in early embryonic zebrafish. CONCLUSIONS: Together, these data provide new insight into ACVR1R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients.

A single cell transcriptome atlas of the developing zebrafish hindbrain.

Segmentation of the vertebrate hindbrain leads to the formation of rhombomeres, each with a distinct anteroposterior identity. Specialised boundary cells form at segment borders that act as a source or regulator of neuronal differentiation. In zebrafish, there is spatial patterning of neurogenesis in which non-neurogenic zones form at boundaries and segment centres, in part mediated by Fgf20 signalling. To further understand the control of neurogenesis, we have carried out single cell RNA sequencing of the zebrafish hindbrain at three different stages of patterning. Analyses of the data reveal known and novel markers of distinct hindbrain segments, of cell types along the dorsoventral axis, and of the transition of progenitors to neuronal differentiation. We find major shifts in the transcriptome of progenitors and of differentiating cells between the different stages analysed. Supervised clustering with markers of boundary cells and segment centres, together with RNA-seq analysis of Fgf-regulated genes, has revealed new candidate regulators of cell differentiation in the hindbrain. These data provide a valuable resource for functional investigations of the patterning of neurogenesis and the transition of progenitors to neuronal differentiation.

Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes.

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, Dmrt3 and Dmrta2 show a medialhigh/laterallow expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to Gsx2 and Pax6 enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.

BMP controls dorsoventral and neural patterning in indirect-developing hemichordates providing insight into a possible origin of chordates.

A defining feature of chordates is the unique presence of a dorsal hollow neural tube that forms by internalization of the ectodermal neural plate specified via inhibition of BMP signaling during gastrulation. While BMP controls dorsoventral (DV) patterning across diverse bilaterians, the BMP-active side is ventral in chordates and dorsal in many other bilaterians. How this phylum-specific DV inversion occurs and whether it is coupled to the emergence of the dorsal neural plate are unknown. Here we explore these questions by investigating an indirect-developing enteropneust from the hemichordate phylum, which together with echinoderms form a sister group of the chordates. We found that in the hemichordate larva, BMP signaling is required for DV patterning and is sufficient to repress neurogenesis. We also found that transient overactivation of BMP signaling during gastrulation concomitantly blocked mouth formation and centralized the nervous system to the ventral ectoderm in both hemichordate and sea urchin larvae. Moreover, this mouthless, neurogenic ventral ectoderm displayed a medial-to-lateral organization similar to that of the chordate neural plate. Thus, indirect-developing deuterostomes use BMP signaling in DV and neural patterning, and an elevated BMP level during gastrulation drives pronounced morphological changes reminiscent of a DV inversion. These findings provide a mechanistic basis to support the hypothesis that an inverse chordate body plan emerged from an indirect-developing ancestor by tinkering with BMP signaling.

The forkhead box containing transcription factor FoxB is a potential component of dorsal-ventral body axis formation in the spider Parasteatoda tepidariorum.

In the spider, determination of the dorsal-ventral body (DV) axis depends on the interplay of the dorsal morphogen encoding gene decapentaplegic (Dpp) and its antagonist, short gastrulation (sog), a gene that is involved in the correct establishment of ventral tissues. Recent work demonstrated that the forkhead domain encoding gene FoxB is involved in dorsal-ventral axis formation in spider limbs. Here, Dpp likely acts as a dorsal morphogen, and FoxB is likely in control of ventral tissues as RNAi-mediated knockdown of FoxB causes dorsalization of the limbs. In this study, we present phenotypes of FoxB knockdown that demonstrate a function in the establishment of the DV body axis. Knockdown of FoxB function leads to embryos with partially duplicated median germ bands (Duplicitas media) that are possibly the result of ectopic activation of Dpp signalling. Another class of phenotypes is characterized by unnaturally slim (dorsal-ventrally compressed) germ bands in which ventral tissue is either not formed, or is specified incorrectly, likely a result of Dpp over-activity. These results suggest that FoxB functions as an antagonist of Dpp signalling during body axis patterning, similarly as it is the case in limb development. FoxB thus represents a general player in the establishment of dorsal-ventral structures during spider ontogeny.

Asymmetric distribution of hypoxia-inducible factor α regulates dorsoventral axis establishment in the early sea urchin embryo.

Hypoxia signaling is an ancient pathway by which animals can respond to low oxygen. Malfunction of this pathway disturbs hypoxic acclimation and can result in various diseases, including cancers. The role of hypoxia signaling in early embryogenesis remains unclear. Here, we show that in the blastula of the sea urchin Strongylocentrotus purpuratus, hypoxia-inducible factor α (HIFα), the downstream transcription factor of the hypoxia pathway, is localized and transcriptionally active on the future dorsal side. This asymmetric distribution is attributable to its oxygen-sensing ability. Manipulations of the HIFα level entrained the dorsoventral axis, as the side with the higher level of HIFα tends to develop into the dorsal side. Gene expression analyses revealed that HIFα restricts the expression of nodal to the ventral side and activates several genes encoding transcription factors on the dorsal side. We also observed that intrinsic hypoxic signals in the early embryos formed a gradient, which was disrupted under hypoxic conditions. Our results reveal an unprecedented role of the hypoxia pathway in animal development.

Vegetally localised Vrtn functions as a novel repressor to modulate bmp2b transcription during dorsoventral patterning in zebrafish.

The vegetal pole cytoplasm represents a crucial source of maternal dorsal determinants for patterning the dorsoventral axis of the early embryo. Removal of the vegetal yolk in the zebrafish fertilised egg before the completion of the first cleavage results in embryonic ventralisation, but removal of this part at the two-cell stage leads to embryonic dorsalisation. How this is achieved remains unknown. Here, we report a novel mode of maternal regulation of BMP signalling during dorsoventral patterning in zebrafish. We identify Vrtn as a novel vegetally localised maternal factor with dorsalising activity and rapid transport towards the animal pole region after fertilisation. Co-injection of vrtn mRNA with vegetal RNAs from different cleavage stages suggests the presence of putative vegetally localised Vrtn antagonists with slower animal pole transport. Thus, vegetal ablation at the two-cell stage could remove most of the Vrtn antagonists, and allows Vrtn to produce the dorsalising effect. Mechanistically, Vrtn binds a bmp2b regulatory sequence and acts as a repressor to inhibit its zygotic transcription. Analysis of maternal-zygotic vrtn mutants further shows that Vrtn is required to constrain excessive bmp2b expression in the margin. Our work unveils a novel maternal mechanism regulating zygotic BMP gradient in dorsoventral patterning.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

DMRT5, DMRT3, and EMX2 Cooperatively Repress Gsx2 at the Pallium-Subpallium Boundary to Maintain Cortical Identity in Dorsal Telencephalic Progenitors.

Specification of dorsoventral regional identity in progenitors of the developing telencephalon is a first pivotal step in the development of the cerebral cortex and basal ganglia. Previously, we demonstrated that the two zinc finger doublesex and mab-3 related (Dmrt) genes, Dmrt5 (Dmrta2) and Dmrt3, which are coexpressed in high caudomedial to low rostrolateral gradients in the cerebral cortical primordium, are separately needed for normal formation of the cortical hem, hippocampus, and caudomedial neocortex. We have now addressed the role of Dmrt3 and Dmrt5 in controlling dorsoventral division of the telencephalon in mice of either sex by comparing the phenotypes of single knock-out (KO) with double KO embryos and by misexpressing Dmrt5 in the ventral telencephalon. We find that DMRT3 and DMRT5 act as critical regulators of progenitor cell dorsoventral identity by repressing ventralizing regulators. Early ventral fate transcriptional regulators expressed in the dorsal lateral ganglionic eminence, such as Gsx2, are upregulated in the dorsal telencephalon of Dmrt3;Dmrt5 double KO embryos and downregulated when ventral telencephalic progenitors express ectopic Dmrt5 Conditional overexpression of Dmrt5 throughout the telencephalon produces gene expression and structural defects that are highly consistent with reduced GSX2 activity. Further, Emx2;Dmrt5 double KO embryos show a phenotype similar to Dmrt3;Dmrt5 double KO embryos, and both DMRT3, DMRT5 and the homeobox transcription factor EMX2 bind to a ventral telencephalon-specific enhancer in the Gsx2 locus. Together, our findings uncover cooperative functions of DMRT3, DMRT5, and EMX2 in dividing dorsal from ventral in the telencephalon.SIGNIFICANCE STATEMENT We identified the DMRT3 and DMRT5 zinc finger transcription factors as novel regulators of dorsoventral patterning in the telencephalon. Our data indicate that they have overlapping functions and compensate for one another. The double, but not the single, knock-out produces a dorsal telencephalon that is ventralized, and olfactory bulb tissue takes over most remaining cortex. Conversely, overexpressing Dmrt5 throughout the telencephalon causes expanded expression of dorsal gene determinants and smaller olfactory bulbs. Furthermore, we show that the homeobox transcription factor EMX2 that is coexpressed with DMRT3 and DMRT5 in cortical progenitors cooperates with them to maintain dorsoventral patterning in the telencephalon. Our study suggests that DMRT3/5 function with EMX2 in positioning the pallial-subpallial boundary by antagonizing the ventral homeobox transcription factor GSX2.

The forkhead transcription factor UNC-130/FOXD integrates both BMP and Notch signaling to regulate dorsoventral patterning of the C. elegans postembryonic mesoderm.

The proper development of a multicellular organism requires precise spatial and temporal coordination of cell intrinsic and cell extrinsic regulatory mechanisms. Both Notch signaling and bone morphogenetic protein (BMP) signaling function to regulate the proper development of the C. elegans postembryonic mesoderm. We have identified the C. elegans FOXD transcription factor UNC-130 as a major target functioning downstream of both BMP signaling and Notch signaling to regulate dorsoventral patterning of the postembryonic mesoderm. We showed that unc-130 expression in the postembryonic M lineage is asymmetric: its absence of expression in the dorsal side of the M lineage requires the antagonism of BMP signaling by the zinc finger transcription factor SMA-9, while its expression in the ventral side of the M lineage is activated by LIN-12/Notch signaling. We further showed that the regulation of UNC-130 expression by BMP signaling and Notch signaling is specific to the M lineage, as the ventral expression of UNC-130 in the embryonically-derived bodywall muscles was not affected in either BMP pathway or Notch pathway mutants. Finally, we showed that the function of UNC-130 in the M lineage is independent of UNC-129, a gene previously shown to function downstream of and be repressed by UNC-130 for axon guidance. Our studies uncovered a new function of UNC-130/FOXD in the C. elegans postembryonic mesoderm, and identify UNC-130 as a critical factor that integrates two independent spatial cues for the proper patterning and fate specification of the C. elegans postembryonic mesoderm.

Kinesin-1 interacts with Bucky ball to form germ cells and is required to pattern the zebrafish body axis.

In animals, specification of the primordial germ cells (PGCs), the stem cells of the germ line, is required to transmit genetic information from one generation to the next. Bucky ball (Buc) is essential for germ plasm (GP) assembly in oocytes, and its overexpression results in excess PGCs in zebrafish embryos. However, the mechanistic basis for the excess PGCs in response to Buc overexpression, and whether endogenous Buc functions during embryogenesis, are unknown. Here, we show that endogenous Buc, like GP and overexpressed Buc-GFP, accumulates at embryonic cleavage furrows. Furthermore, we show that the maternally expressed zebrafish Kinesin-1 Kif5Ba is a binding partner of Buc and that maternal kif5Ba (Mkif5Ba) plays an essential role in germline specification in vivo. Specifically, Mkif5Ba is required to recruit GP to cleavage furrows and thereby specifies PGCs. Moreover, Mkif5Ba is required to enrich Buc at cleavage furrows and for the ability of Buc to promote excess PGCs, providing mechanistic insight into how Buc functions to assemble embryonic GP. In addition, we show that Mkif5Ba is also essential for dorsoventral (DV) patterning. Specifically, Mkif5Ba promotes formation of the parallel vegetal microtubule array required to asymmetrically position dorsal determinants (DDs) towards the prospective dorsal side. Interestingly, whereas Syntabulin and wnt8a translocation depend on kif5Ba, grip2a translocation does not, providing evidence for two distinct mechanisms by which DDs might be asymmetrically distributed. These studies identify essential roles for maternal Kif5Ba in PGC specification and DV patterning, and provide mechanistic insight into Buc functions during early embryogenesis.

Extramacrochaetae functions in dorsal-ventral patterning of Drosophila imaginal discs.

One of the seminal events in the history of a tissue is the establishment of the anterior-posterior, dorsal-ventral (D/V) and proximal-distal axes. Axis formation is important for the regional specification of a tissue and allows cells along the different axes to obtain directional and positional information. Within the Drosophila retina, D/V axis formation is essential to ensure that each unit eye first adopts the proper chiral form and then rotates precisely 90° in the correct direction. These two steps are important because the photoreceptor array must be correctly aligned with the neurons of the optic lobe. Defects in chirality and/or ommatidial rotation will lead to disorganization of the photoreceptor array, misalignment of retinal and optic lobe neurons, and loss of visual acuity. Loss of the helix-loop-helix protein Extramacrochaetae (Emc) leads to defects in both ommatidial chirality and rotation. Here, we describe a new role for emc in eye development in patterning the D/V axis. We show that the juxtaposition of dorsal and ventral fated tissue in the eye leads to an enrichment of emc expression at the D/V midline. emc expression at the midline can be eliminated when D/V patterning is disrupted and can be induced in situations in which ectopic boundaries are artificially generated. We also show that emc functions downstream of Notch signaling to maintain the expression of four-jointed along the midline.

Zic Genes in Teleosts: Their Roles in Dorsoventral Patterning in the Somite.

The medaka contains seven zic genes, two of which, zic1 and zic4, have been studied extensively. The analyses are mainly based on the double anal fin (Da) mutant, which was isolated from the wild. Da is an enhancer mutant of zic1/zic4, and the expression of zic1/zic4 is specifically lost in the dorsal half of the somites, which leads to a mirror-image duplication of the ventral half across the lateral midline from larva to adult. The studies of medaka Da give us important insights into the function of zic1/zic4 in mesodermal tissues and also the mechanism of dorsoventral patterning in the vertebrate trunk region occurring during late development, which is a long-standing mystery in developmental biology. In this chapter, we introduce genomic organization of medaka zic genes and discuss their function, mainly focusing on zic1 and zic4 in dorsoventral patterning of the trunk region and possible connections to human congenital disorders.

Vertebrate Axial Patterning: From Egg to Asymmetry.

The emergence of the bilateral embryonic body axis from a symmetrical egg has been a long-standing question in developmental biology. Historical and modern experiments point to an initial symmetry-breaking event leading to localized Wnt and Nodal growth factor signaling and subsequent induction and formation of a self-regulating dorsal "organizer." This organizer forms at the site of notochord cell internalization and expresses primarily Bone Morphogenetic Protein (BMP) growth factor antagonists that establish a spatiotemporal gradient of BMP signaling across the embryo, directing initial cell differentiation and morphogenesis. Although the basics of this model have been known for some time, many of the molecular and cellular details have only recently been elucidated and the extent that these events remain conserved throughout vertebrate evolution remains unclear. This chapter summarizes historical perspectives as well as recent molecular and genetic advances regarding: (1) the mechanisms that regulate symmetry-breaking in the vertebrate egg and early embryo, (2) the pathways that are activated by these events, in particular the Wnt pathway, and the role of these pathways in the formation and function of the organizer, and (3) how these pathways also mediate anteroposterior patterning and axial morphogenesis. Emphasis is placed on comparative aspects of the egg-to-embryo transition across vertebrates and their evolution. The future prospects for work regarding self-organization and gene regulatory networks in the context of early axis formation are also discussed.

Multiscale modeling of dorsoventral patterning in Drosophila.

The role of mathematical models of signaling networks is showcased by examples from Drosophila development. Three models of consecutive stages in dorsoventral patterning are presented. We begin with a compartmental model of intracellular reactions that generates a gradient of nuclear-localized Dorsal, exhibiting constant shape and dynamic amplitude. A simple thermodynamic model of equilibrium binding explains how a spatially uniform transcription factor, Zelda, can act in combination with a graded factor, Dorsal, to cooperatively regulate gene expression borders. Finally, we formulate a dynamic and stochastic model that predicts spatiotemporal patterns of Sog expression based on known patterns of its transcription factor, Dorsal. The future of coupling multifarious models across multiple temporal and spatial scales is discussed.

prdm12b specifies the p1 progenitor domain and reveals a role for V1 interneurons in swim movements.

Proper functioning of the vertebrate central nervous system requires the precise positioning of many neuronal cell types. This positioning is established during early embryogenesis when gene regulatory networks pattern the neural tube along its anteroposterior and dorsoventral axes. Dorsoventral patterning of the embryonic neural tube gives rise to multiple progenitor cell domains that go on to differentiate unique classes of neurons and glia. While the genetic program is reasonably well understood for some lineages, such as ventrally derived motor neurons and glia, other lineages are much less characterized. Here we show that prdm12b, a member of the PR domain containing-family of transcriptional regulators, is expressed in the p1 progenitor domain of the zebrafish neural tube in response to Sonic Hedgehog signaling. We find that disruption of prdm12b function leads to dorsal expansion of nkx6.1 expression and loss of p1-derived eng1b-expressing V1 interneurons, while the adjacent p0 and p2 domains are unaffected. We also demonstrate that prdm12b-deficient fish exhibit an abnormal touch-evoked escape response with excessive body contractions and a prolonged response time, as well as an inability to coordinate swimming movements, thereby revealing a functional role for V1 interneurons in locomotor circuits. We conclude that prdm12b is required for V1 interneuron specification and that these neurons control swimming movements in zebrafish.

Rnf220 is Implicated in the Dorsoventral Patterning of the Hindbrain Neural Tube in Mice.

Rnf220 is reported to regulate the patterning of the ventral spinal neural tube in mice. The brainstem has divergent connections with peripheral and central targets and contains unique internal neuronal groups, but the role of Rnf220 in the early development of the hindbrain has not been explored. In this study, Nestin-Cre-mediated conditional knockout (Rnf220 Nestin CKO) mice were used to examine if Rnf220 is involved in the early morphogenesis of the hindbrain. Rnf220 showed restricted expression in the ventral half of ventricular zone (VZ) of the hindbrain at embryonic day (E) 10.5, and as development progressed, Rnf220-expressing cells were also present in the mantle zone outside the VZ at E12.5. In Rnf220 Nestin CKO embryos, alterations of progenitor domains in the ventral VZ were observed at E10.5. There were significant reductions of the p1 and p2 domains shown by expression of Dbx1, Olig2, and Nkx6.1, accompanied by a ventral expansion of the Dbx1+ p0 domain and a dorsal expansion of the Nkx2.2+ p3 domain. Different from the case in the spinal cord, the Olig2+ pMN (progenitors of somatic motor neuron) domain shifted and expanded dorsally. Notably, the total range of the ventral VZ and the extent of the dorsal tube were unchanged. In addition, the post-mitotic cells derived from their corresponding progenitor domain, including oligodendrocyte precursor cells (OPCs) and serotonergic neurons (5-HTNs), were also changed in the same trend as the progenitor domains do in the CKO embryos at E12.5. In summary, our data suggest similar functions of Rnf220 in the hindbrain dorsoventral (DV) patterning as in the spinal cord with different effects on the pMN domain. Our work also reveals novel roles of Rnf220 in the development of 5-HTNs and OPCs.

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.

BMP Signaling: Lighting up the Way for Embryonic Dorsoventral Patterning.

One of the most significant events during early embryonic development is the establishment of a basic embryonic body plan, which is defined by anteroposterior, dorsoventral (DV), and left-right axes. It is well-known that the morphogen gradient created by BMP signaling activity is crucial for DV axis patterning across a diverse set of vertebrates. The regulation of BMP signaling during DV patterning has been strongly conserved across evolution. This is a remarkable regulatory and evolutionary feat, as the BMP gradient has been maintained despite the tremendous variation in embryonic size and shape across species. Interestingly, the embryonic DV axis exhibits robust stability, even in face of variations in BMP signaling. Multiple lines of genetic, molecular, and embryological evidence have suggested that numerous BMP signaling components and their attendant regulators act in concert to shape the developing DV axis. In this review, we summarize the current knowledge of the function and regulation of BMP signaling in DV patterning. Throughout, we focus specifically on popular model animals, such as Xenopus and zebrafish, highlighting the similarities and differences of the regulatory networks between species. We also review recent advances regarding the molecular nature of DV patterning, including the initiation of the DV axis, the formation of the BMP gradient, and the regulatory molecular mechanisms behind BMP signaling during the establishment of the DV axis. Collectively, this review will help clarify our current understanding of the molecular nature of DV axis formation.