Zone Of Polarizing Activity Regulatory Sequence Research Articles

Caecilian Genomes Reveal the Molecular Basis of Adaptation and Convergent Evolution of Limblessness in Snakes and Caecilians.

We present genome sequences for the caecilians Geotrypetes seraphini (3.8 Gb) and Microcaecilia unicolor (4.7 Gb), representatives of a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. More than 69% of both genomes are composed of repeats, with retrotransposons being the most abundant. We identify 1,150 orthogroups that are unique to caecilians and enriched for functions in olfaction and detection of chemical signals. There are 379 orthogroups with signatures of positive selection on caecilian lineages with roles in organ development and morphogenesis, sensory perception, and immunity amongst others. We discover that caecilian genomes are missing the zone of polarizing activity regulatorysequence (ZRS) enhancer of Sonic Hedgehog which is also mutated in snakes. In vivo deletions have shown ZRS is required for limb development in mice, thus, revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.

The pZRS non-coding regulatory mutation resulting in triphalangeal thumb-polysyndactyly syndrome changes the pattern of local interactions.

Herein, we report on a large Polish family presenting with a classical triphalangeal thumb-polysyndactyly syndrome (TPT-PS). This rare congenital limb anomaly is generally caused by microduplications encompassing the Sonic Hedgehog (SHH) limb enhancer, termed the zone of polarizing activity (ZPA) regulatory sequence (ZRS). Recently, a pathogenic variant in the pre-ZRS (pZRS), a conserved sequence located near the ZRS, has been described in a TPT-PS Dutch family. We performed targeted ZRS sequencing, array comparative genomic hybridization, and whole-exome sequencing. Next, we sequenced the recently described pZRS region. Finally, we performed a circular chromatin conformation capture-sequencing (4C-seq) assay on skin fibroblasts of one affected family member and control samples to examine potential alterations in the SHH regulatory domain and functionally characterize the identified variant. We found that all affected individuals shared a recently identified pathogenic point mutation in the pZRS region: NC_000007.14:g.156792782C>G (GRCh38/hg38), which is the same as in the Dutch family. The results of 4C-seq experiments revealed increased interactions within the whole SHH regulatory domain (SHH-LMBR1 TAD) in the patient compared to controls. Our study expands the number of TPT-PS families carrying a pathogenic alteration of the pZRS and underlines the importance of routine pZRS sequencing in the genetic diagnostics of patients with TPT-PS or similar phenotypes. The pathogenic mutation causative for TPT-PS in our patient gave rise to increased interactions within the SHH regulatory domain in yet unknown mechanism.

An E‐Box Binding Site is Sufficient for ZPA Regulatory Sequence (ZRS) Activity in the Limb

The zone of polarizing activity (ZPA) is a cluster of mesodermal cells that secrete Sonic hedgehog (Shh) to regulate anterior‐posterior patterning during limb development. The ZPA regulatory sequence (ZRS) is the limb‐specific enhancer that controls Shh expression. ZRS microdeletion abrogates Shh expression and posterior limb development, whereas ZRS single nucleotide variants (SNVs) produce anterior ectopic Shh and preaxial polydactyly. However, the ZRS mechanism of action is not well characterized. Transcription factors (TFs) Hand2, Twist1, and Hoxd13 regulate Shh expression and ZRS contains putative binding sites for each: two Hoxd13 binding sites and three E‐boxes, sites targeted by basic helix‐loop‐helix (bHLH) TFs such as Hand2 and Twist1. Further, reports suggest these TFs may act through the ZRS: Hand2 purportedly binds the ZRS through a central E‐box; as bHLH‐TFs Hand2 and Twist1 may regulate ZRS as homo‐ or heterodimers; and both Hand2 and Hoxd13 activate the ZRS in vitro. Previously we demonstrated that disrupting all five binding sites eliminates ZRS activity. In this report, we further investigate these sites to determine the contribution of each to ZRS activity. We hypothesized that the Hand2 binding site is required for activity and that other E‐boxes and Hoxd13 sites localize ZRS activity to the distal posterior limb mesoderm.We assessed ZRS activity via an enhancer‐GFP reporter, altering all five binding sites to eliminate activity, then reinstating each site individually using site‐directed mutagenesis. Constructs were electroporated into the presumptive limbs of chicken embryos (Hamburger‐Hamilton stage 14). Enhancer activity was evaluated 48 hours post‐electroporation by fluorescence microscopy. Restoring either Hoxd13 site led to weak, focal activity. Restoring two of the E‐boxes had little to no effect, however, restoring the third, 3’ E‐box was sufficient for robust activity. This was unexpected since the central E‐box was reported as the likely Hand2 binding site. Our data suggest that the 3’ E‐box is likely the critical site used by Hand2 in ZRS‐mediated Shh regulation. Additionally, the Hoxd13 binding sites appear to impart focal activity supporting a role for localization. These findings further characterize the mechanism used to regulate focal Shh expression during limb development.

LHX2 Regulates Shh Expression in the Limb Independent of the ZPA Regulatory Sequence

During limb development, proximal‐distal patterning is controlled by fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER), while anterior‐posterior expansion is coordinated by sonic hedgehog (SHH) released from the zone of polarizing activity (ZPA). Both FGFs and SHH regulate each other through an autoregulatory loop. The transcription factor LIM homeodomain 2 (LHX2) is an intermediate of the Fgf‐to‐Shh portion of the loop. In the absence of Lhx2 (and its redundant family member, Lhx9), Shh expression is markedly reduced. However, the mechanism by which LHX2 mediates FGF‐regulation of Shh is unclear. Shh is regulated by a limb‐specific enhancer called the ZPA Regulatory Sequence (ZRS). Using in silicoanalysis, our lab has identified two putative LHX2 binding sites within the ZRS. We hypothesized that LHX2 regulates Shh expression directly via the ZRS. To determine if LHX2 binding sites were necessary for ZRS activity, we mutated both LHX2 binding sites within a control tk‐ZRS‐eGFP reporter construct using site‐directed mutagenesis. We electroporated these constructs into the presumptive limb buds of chicken embryos (Hamburger‐Hamilton stage 14) and assessed for changes in ZRS activity in the limb buds 48 hours post transfection via fluorescence microscopy. We found that the mutation of both LHX2 binding sites within the ZRS resulted in activity similar to our control. These results suggest that LHX2 may not bind directly to the ZRS in its regulation of Shh. Instead, LHX2 may use another pathway to regulate Shhexpression. Further experiments are needed to confirm this connection. Investigating the mechanism by which LHX2 regulates Shh will provide insight into how LHX2 contributes to coordinated patterned‐limb development.

ZPA Regulatory Sequence (ZRS) Activity in the Limb is Eliminated by Loss of Hand2, Twist1, and Hoxd13 Binding Sites

During limb development a cluster of mesodermal cells in the distal posterior limb bud, the zone of polarizing activity (ZPA), regulates patterning along the anterior-posterior axis. The signaling molecule responsible for this patterning is Sonic hedgehog (Shh), which is controlled by a highly conserved limb-specific enhancer, the ZPA regulatory sequence (ZRS).ZRS microdeletion results in limbs that fail to express Shh and do not develop posterior limb elements, whereas many ZRS single nucleotide variants (SNVs) lead to anterior ectopic Shh expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. The ZRS contains putative binding sites for Hand2, Twist1, and Hoxd13: transcription factors (TFs) known to regulate Shh. Both Hand2 and Twist1 are basic helix-loop-helix (bHLH) TFs and are thought to function as homo- or heterodimers, while Hoxd13 has been shown to interact with Hand2.The present study's objective is to determine the role of these TFs in ZRS function. We hypothesize that altering Hand2, Twist1, and Hoxd13 bindings sites, either alone or in concert, will disrupt ZRS activity. To evaluate enhancer activity, ZRS was cloned into an enhancer-GFP reporter construct. Hand2, Twist1, and Hoxd13 binding sites were altered using site-directed mutagenesis, and constructs were electroporated into the presumptive limb buds of chicken embryos (Hamburger-Hamilton stage 14). GFP expression was evaluated 48 hours post-electroporation. We determined that mutating the binding sites for all three TFs in concert resulted in loss of ZRS activity. However, constructs with either Twist1 or Hoxd13 binding sites intact remained active. These data suggest that Twist1 and Hoxd13 may have either redundant or temporally distinct roles in ZRS regulation of Shh. Future work will determine whether these TFs bind the ZRS at the specific sites we have identified.

The ZPA Regulatory Sequence (ZRS) Hand2 and Twist1 Binding Sites are Not Necessary for ZRS Activity

Anterior-posterior patterning in the developing limb is regulated by Sonic Hedgehog (Shh), which is expressed in the Zone of Polarizing Activity (ZPA). Shh is regulated by a limb-specific enhancer known as the ZPA Regulatory Sequence (ZRS). Mutations in the highly conserved ZRS have been linked to limb malformations in humans. Single nucleotide variants (SNVs) cause digit duplications, such as preaxial polydactyly (PPD) and triphalangeal thumb-polysyndactyly syndrome (TPTPS). The ZRS mechanism is not well understood. Conditional knockout of Hand2 results in a delta-Shh phenotype, indicating the importance of Hand2 in Shh regulation. Hand2 and Twist1 are basic helix-loop-helix (bHLH) transcription factors, capable of homo- and hetero-dimerization, that bind E-boxes (CANNTG) with some promiscuity. The objective of the present study was to determine whether the three ZRS E-boxes are necessary for activity. We hypothesize that altering E-boxes will disrupt ZRS function. Enhancer activity was evaluated using a ZRS-GFP reporter plasmid. We generated a construct with all three E-boxes altered using site-directed mutagenesis. Constructs were electroporated into presumptive forelimb buds in Hamburger-Hamilton stage 14 chicken embryos. Enhancer activity was recorded using fluorescent microscopy. Surprisingly, modification of the Hand2 and Twist1 binding sites did not affect ZRS activity. Future work is needed to identify what transcription factors are indispensable for ZRS regulation of Shh.

Loss of HAND2 and HOXD13 Binding Sites in the ZPA Regulatory Sequence (ZRS) is not Sufficient to Disrupt Activity in the Limb

The ZPA regulatory sequence (ZRS) is a limb‐specific enhancer that localizes Sonic Hedgehog (SHH) expression to the zone of polarizing activity (ZPA), which consequently regulates anteroposterior patterning in the developing limb. ZRS deletion lacks SHH expression in the limb resulting in loss of posterior limb structures, while ZRS single nucleotide variants (SNVs) can lead to ectopic SHH expression causing preaxial polydactyly (PPD). However, the ZRS mechanism of action is not well characterized. HAND2 and HOXD13 are key transcription factors in SHH regulation that bind to the ZRS, potentially modulating SHH transcription. The present study’s objective was to determine the role of HAND2 and HOXD13 binding sites in ZRS function. To evaluate ZRS enhancer activity, ZRS was cloned into an enhancer‐GFP reporter construct. HAND2 and HOXD13 binding sites were altered using site‐directed mutagenesis, and contructs were electroporated into presumptive limb buds in Hamburger‐Hamilton stage 14 chicken embryos. GFP expression was evaluated 48 hours post‐electroporation. ZRS with mutated HAND2 and HOXD13 binding sites retained activity in the limb bud in a pattern that overlapped ZRS activity. This is unexpected since HAND2 and HOXD13 are known to be critical for SHH expression. Future work is needed to clarify the binding site(s) critical to activity and to characterize the factors involved in ZRS function.Support or Funding InformationSupported in part by a grant from the Department of Pathology, Loma Linda University.

Genetic heterogeneity in type III familial cutaneous syndactyly and linkage to chromosome 7q36

The ZRS (zone of polarizing activity regulatory sequence) is a long-range limb-specific Sonic Hedgehog (SHH) enhancer. In humans, the ZRS is located in chromosome 7q36 within intron 5 of LMBR1; approximately 1 Mb telomeric of SHH. Point mutations and duplications of the ZRS lead to a variable phenotype of preaxial polydactyly/triphalangeal thumb, tibial hypoplasia, radial ray deficiency, and type IV familial syndactyly (syndactyly of all digits with polydactyly). The ZRS is conserved among mammals and fish and regulates the expression of SHH. In mice, the conserved ZRS within the Lmbr1 gene is found in chromosome 5. The Hammertoe (Hm) mouse mutants have a mutation in the Lmbr1 locus and show syndactyly of digits 2-5 without polydactyly. No previous reports have described isolated syndactyly without polydactyly to be related to the LMBR1 locus in humans. In this report, we describe a family with simple cutaneous syndactyly involving digits 2-5, without polydactyly which is consistent with the phenotype of type III syndactyly. The locus we identified on ch7q36.3 is syntenic to the Hm locus; and affected members of the family had a phenotype analogous to Hm. Hence, the type of syndactyly described in the current report may be equivalent to Hm mice.

A novel mutation in the SHH long‐range regulator (ZRS) is associated with preaxial polydactyly, triphalangeal thumb, and severe radial ray deficiency

Sonic Hedgehog (SHH) within the posteriorly located zone of polarizing activity is the main controller of the antero-posterior axis of limb development. The ZRS (zone of polarizing activity regulatory sequence) is a long-range limb-specific SHH enhancer. Several point mutations in the ZRS have been described in humans. These mutations cause enhanced SHH activity and ectopic anterior expression of SHH and a variable phenotype of preaxial polydactyly and triphalangeal thumb. Absent thumb or radius has not been reported with ZRS mutations. Here, we report on a family with a variable phenotype of preaxial polydactyly as well as absent thumb and radius, with kidney and cardiac defects. The family was screened for SALL1, SALL4, and TBX5 mutations, but all were normal. Finally, they were screened for ZRS mutations, which showed a novel point mutation within the ZRS, NG_009240.1: g.106954C>T (traditional nomenclature: ZRS619C>T) in the five affected members. This mutation was not previously reported in any public domain database, and was not found in our healthy and ethnically matched control individuals or unaffected family members. We hypothesize that interactions of SHH and SALL1 explain the overlapping features of the family described here and patients with Townes-Brocks syndrome.

Highlights in DD

“Highlights” calls attention to exciting advances in developmental biology that have recently been reported in Developmental Dynamics. Development is a broad field encompassing many important areas. To reflect this fact, the section spotlights significant discoveries that occur across the entire spectrum of developmental events and problems: from new experimental approaches, to novel interpretations of results, to noteworthy findings utilizing different developmental organisms. Moving on (Evolution of Vertebrate Appendicular Structures: Insight From Genetic and Palaeontological Data by Amir Ali Abbasi, Dev Dyn 240:1005–1016). How fins evolved into limbs is one of the quintessential examples of morphological evolution, the study of the diversification of body form over time. In this Special Issue on limb development, Abbasi assembles information from molecular biology, genetics, comparative biology, and paleontology to illustrate key morphological and molecular events that enact this grand change. One remaining mystery is how the distal bones of a primordial fin/limb transformed into modern toes and fingers. Chipping away at the problem, he describes how certain molecular pathways (i.e., Shh, 5′Hoxd cluster) are involved in fin/limb patterning both pre- and postevolutionary digit formation. He further explains how the morphological advancement could be triggered by molecular changes in cis-regulatory elements of conserved genes that alter sites and/or timing of activity. His careful analysis of what is known helps him to pinpoint specific areas of research that could help fill the morphological gap. These insights should enable researchers to hit the ground running. Tales of the pinkie (HMGB Factors Are Required for Posterior Digit Development Through Integrating Signaling Pathway Activities by Junji Itou, Noboru Taniguchi, Isao Oishi, Hiroko Kawakami, Martin Lotz, Yasuhiko Kawakami, Dev Dyn 240:1151–1162) Hmgb1−/−;Hmbg2+/− mice have a subtle forelimb phenotype, loss of the most posterior digit (analogous to the human pinkie finger). Although the defect may seem minor, it speaks volumes about the role of these chromatin factors in limb patterning. The phenotype mimics reduced signaling of Sonic Hedgehog (Shh) in the limb bud, and Itou et. al, verify that Hmgb1−/−;Hmbg2+/− also have this molecular defect. How, then, might HMGB factors regulate limb Shh? The authors suspect the interaction is indirect. The largely redundant HMGB1 and 2 bind DNA nonsequence specifically and alter chromatin architecture, and also facilitate Wnt/β-catenin signaling. In light of the latter finding, they are surprised to find that dorsal ectoderm Wnt7a signaling, which regulates posterior Shh, is normal in Hmgb1−/−;Hmbg2+/− limbs. They do, however, find that Wnt/β-catenin targets display reduced expression in the posterior mesenchyme, suggesting the tissue is an uncharacterized region of Wnt7a signaling. Supporting this idea, Hmbg1 and 2 also facilitates Wnt/β-catenin signaling in numerous tissues in the developing zebrafish, including the pectoral fin, a forelimb homolog. In addition to a role in Wnt/β-catenin signaling, there is also evidence that HMGB factors modulate BMP signaling. The authors discuss how proper development of the little finger may hinge on integration of these pathways by HMGB 1 and 2. Golden eggs (Identification of Spontaneous Mutations Within the Long-Range Limb-Specific Sonic Hedgehog Enhancer (ZRS) That Alter Sonic Hedgehog Expression in the Chicken Limb Mutants oligozeugodactyly and Silkie Breed by Sarah A. Maas, Takayuki Suzuki, and John F. Fallon, Dev Dyn 240:1212–1222) What might be considered an oddity to an animal breeder can be a goldmine to a biologist. So it is with the spontaneous oligozeugodactyly (ozd) chicken mutant, which lacks all but one digit on each foot, and the preaxial polydactylous (PPD) Silke breed with extra digits. Because mutations in the evolutionarily conserved zone of polarizing activity (ZPA) regulatory sequence (ZRS)—which controls posterior limb Shh expression and anteroposterior limb patterning—has been implicated in human PPD, Maas et al. examined the ZRS in the two strains. Eureka! While ozd chickens lack most of the ZRS enhancer, the Silkie ZRS has a single point mutation within it. To further investigate ZRS function, the group devised the first enhancer assay that works in the developing limb bud in vivo. They find that although the ozd mutant does not express Shh, the wild-type ZRS enhancer drives expression of β-galactosidase (β-gal) in a small group of cells within the pre-ZPA, suggesting they are still ZPA competent. The Silkie ZRS sequence drives β-gal expression that is expanded to the anterior domain, mirroring the ectopic Shh expression observed in this breed. Together the results suggest the ZRS receives both positive and negative input. The next pot of gold goes to the one who determines how factors that prepattern the limb act upon the ZRS to fine-tune Shh expression.

Identification of spontaneous mutations within the long‐range limb‐specific Sonic hedgehog enhancer (ZRS) that alter Sonic hedgehog expression in the chicken limb mutants oligozeugodactyly and silkie breed

The evolutionarily conserved, non-coding ~800-base-pair (bp) zone of polarizing activity (ZPA) regulatory sequence (ZRS) controls Shh expression in the posterior limb. We report that the chicken mutant oligozeugodactyly (ozd), which lacks limb Shh expression, has a large deletion within the ZRS. Furthermore, the preaxial polydactylous, Silkie Breed chicken, which develops ectopic anterior limb Shh expression, has a single bp change within the ZRS. Using an in vivo reporter assay to examine enhancer function in the chick limb, we demonstrate that the wild-type ZRS drives β-galactosidase reporter expression in the ZPA of both wild-type and ozd limbs. The Silkie ZRS drives β-galactosidase in both posterior and anterior Shh domains in wild-type limb buds. These results support the hypothesis that the ZRS integrates positive and negative prepatterned regulatory inputs in the chicken model system and demonstrate the utility of the chicken limb as an efficient genetic system for gene regulatory studies.

Preaxial polydactyly/triphalangeal thumb is associated with changed transcription factor-binding affinity in a family with a novel point mutation in the long-range cis-regulatory element ZRS

A cis-regulatory sequence also known as zone of polarizing activity (ZPA) regulatory sequence (ZRS) located in intron 5 of LMBR1 is essential for expression of sonic hedgehog (SHH) in the developing posterior limb bud mesenchyme. Even though many point mutations causing preaxial duplication defects have been reported in ZRS, the underlying regulatory mechanism is still unknown. In this study, we analyzed the effect on transcription factor binding of a novel ZRS point mutation (463T>G) in a Pakistani family with preaxial polydactyly and triphalangeal thumb. Electrophoretical mobility shift assay demonstrated a marked difference between wild-type and the mutant probe, which uniquely bound one or several transcription factors extracted from Caco-2 cells. This finding supports a model in which ectopic anterior SHH expression in the developing limb results from abnormal binding of one or more transcription factors to the mutant sequence.