A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs.

Spatial and Quantitative Detection of BMP Activity in Mouse Embryonic Limb Buds.

Modulation of bone morphogenetic protein (BMP) activity is essential to the progression of limb development in the mouse embryo. Genetic disruption of BMP signaling at various stages of limb development causes defects ranging from complete limb agenesis to oligodactyly, polydactyly, webbing, and chondrodysplasia. To probe the state of BMP signaling in early limb buds, we designed two sets of primers to measure both spatially and quantitatively the transcription of nine key genes indicative of canonical BMP activity. One set is used to generate digoxigenin (DIG)-labeled antisense RNA probes for whole-mount mRNA in situ hybridization, while the second set is used for SYBR® Green-based quantitative PCR on limb bud cDNA. Here we describe step-by-step protocols for both methods around this specific set of genes.

Cellular heterogeneity in the ureteric progenitor niche and distinct profiles of branching morphogenesis in organ development.

Branching morphogenesis creates arborized epithelial networks. In the mammalian kidney, an epithelial progenitor pool at ureteric branch tips (UBTs) creates the urine-transporting collecting system. Using region-specific mouse reporter strains, we performed an RNA-seq screen, identifying tip- and stalk-enriched gene sets in the developing collecting duct system. Detailed in situ hybridization studies of tip-enriched predictions identified UBT-enriched gene sets conserved between the mouse and human kidney. Comparative spatial analysis of their UBT niche expression highlighted distinct patterns of gene expression revealing novel molecular heterogeneity within the UBT progenitor population. To identify kidney-specific and shared programs of branching morphogenesis, comparative expression studies on the developing mouse lung were combined with in silico analysis of the developing mouse salivary gland. These studies highlight a shared gene set with multi-organ tip enrichment and a gene set specific to UBTs. This comprehensive analysis extends our current understanding of the ureteric branch tip niche.

Differential regulation of mouse and human nephron progenitors by the Six family of transcriptional regulators.

Nephron endowment is determined by the self-renewal and induction of a nephron progenitor pool established at the onset of kidney development. In the mouse, the related transcriptional regulators Six1 and Six2 play non-overlapping roles in nephron progenitors. Transient Six1 activity prefigures, and is essential for, active nephrogenesis. By contrast, Six2 maintains later progenitor self-renewal from the onset of nephrogenesis. We compared the regulatory actions of Six2 in mouse and human nephron progenitors by chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq). Surprisingly, SIX1 was identified as a SIX2 target unique to the human nephron progenitors. Furthermore, RNA-seq and immunostaining revealed overlapping SIX1 and SIX2 activity in 16 week human fetal nephron progenitors. Comparative bioinformatic analysis of human SIX1 and SIX2 ChIP-seq showed each factor targeted a similar set of cis-regulatory modules binding an identical target recognition motif. In contrast to the mouse where Six2 binds its own enhancers but does not interact with DNA around Six1, both human SIX1 and SIX2 bind homologous SIX2 enhancers and putative enhancers positioned around SIX1. Transgenic analysis of a putative human SIX1 enhancer in the mouse revealed a transient, mouse-like, pre-nephrogenic, Six1 regulatory pattern. Together, these data demonstrate a divergence in SIX-factor regulation between mouse and human nephron progenitors. In the human, an auto/cross-regulatory loop drives continued SIX1 and SIX2 expression during active nephrogenesis. By contrast, the mouse establishes only an auto-regulatory Six2 loop. These data suggest differential SIX-factor regulation might have contributed to species differences in nephron progenitor programs such as the duration of nephrogenesis and the final nephron count.

ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling.

Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here, we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis, we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25(ENU)). Unlike Vps25-null embryos we generated, Vps25(ENU/ENU) mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyperactivation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25(ENU/ENU) Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however, SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning.

Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds.

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2-Cre was used to conditionally inactivate Smad4 in the ectoderm of fore- and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo- and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial-mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER-FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling.

Smad4 is required to induce digit ray primordia and to initiate the aggregation and differentiation of chondrogenic progenitors in mouse limb buds.

SMAD4 is an essential mediator of canonical TGFß/BMP signal transduction and we inactivated Smad4 in mouse limb buds from early stages onward to study its functions in the mesenchyme. While this Smad4 inactivation did not alter the early Sox9 distribution, prefiguring the chondrogenic primordia of the stylopod and zeugopod, it disrupted formation of all Sox9-positive digit ray primordia. Specific inactivation of Smad4 during handplate development pointed to its differential requirement for posterior and anterior digit ray primordia. At the cellular level, Smad4 deficiency blocked the aggregation of Sox9-positive progenitors, thereby preventing chondrogenic differentiation as revealed by absence of collagen type II. The progressive loss of SOX9 due to disrupting digit ray primordia and chondrogenesis was paralleled by alterations in genes marking other lineages. This pointed to a general loss of tissue organization and diversion of mutant cells toward non-specific connective tissue. Conditional inactivation of Bmp2 and Bmp4 indicated that the loss of digit ray primordia and increase in connective tissue were predominantly a consequence of disrupting SMAD4-mediated BMP signal transduction. In summary, our analysis reveals that SMAD4 is required to initiate: (1) formation of the Sox9-positive digit ray primordia; and (2) aggregation and chondrogenic differentiation of all limb skeletal elements.

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.

A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning.

Embryogenesis depends on self-regulatory interactions between spatially separated signaling centers, but few of these are well understood. Limb development is regulated by epithelial-mesenchymal (e-m) feedback loops between sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist Gremlin1 (GREM1). By combining mouse molecular genetics with mathematical modeling, we showed that BMP4 first initiates and SHH then propagates e-m feedback signaling through differential transcriptional regulation of Grem1 to control digit specification. This switch occurs by linking a fast BMP4/GREM1 module to the slower SHH/GREM1/FGF e-m feedback loop. This self-regulatory signaling network results in robust regulation of distal limb development that is able to compensate for variations by interconnectivity among the three signaling pathways.

Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system.

A wealth of classical embryological manipulation experiments taking mainly advantage of the chicken limb buds identified the apical ectodermal ridge (AER) and the zone of polarizing activity (ZPA) as the respective ectodermal and mesenchymal key signaling centers coordinating proximodistal (PD) and anteroposterior (AP) limb axis development. These experiments inspired Wolpert's French flag model, which is a classic among morphogen gradient models. Subsequent molecular and genetic analysis in the mouse identified retinoic acid as proximal signal, and fibroblast growth factors (FGFs) and sonic hedgehog (SHH) as the essential instructive signals produced by AER and ZPA, respectively. Recent studies provide good evidence that progenitors are specified early with respect to their PD and AP fates and that morpho-regulatory signaling is also required for subsequent proliferative expansion of the specified progenitor pools. The determination of particular fates seems to occur rather late and depends on additional signals such as bone morphogenetic proteins (BMPs), which indicates that cells integrate signaling inputs over time and space. The coordinate regulation of PD and AP axis patterning is controlled by an epithelial-mesenchymal feedback signaling system, in which transcriptional regulation of the BMP antagonist Gremlin1 integrates inputs from the BMP, SHH, and FGF pathways. Vertebrate limb-bud development is controlled by a 4-dimensional (4D) patterning system integrating positive and negative regulatory feedback loops, rather than thresholds set by morphogen gradients.