Ramón A. Espinoza-Lewis

Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development

Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.

Shox2 is essential for the differentiation of cardiac pacemaker cells by repressing Nkx2-5

The pacemaker is composed of specialized cardiomyocytes located within the sinoatrial node (SAN), and is responsible for originating and regulating the heart beat. Recent advances towards understanding the SAN development have been made on the genetic control and gene interaction within this structure. Here we report that the Shox2 homeodomain transcription factor is restrictedly expressed in the sinus venosus region including the SAN and the sinus valves during embryonic heart development. Shox2 null mutation results in embryonic lethality due to cardiovascular defects, including an abnormal low heart beat rate (bradycardia) and severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation. Genetically, the lack of Tbx3 and Hcn4 expression, along with ectopic activation of Nppa, Cx40, and Nkx2-5 in the Shox2(-/-) SAN region, indicates a failure in SAN differentiation. Furthermore, Shox2 overexpression in Xenopus embryos results in extensive repression of Nkx2-5 in the developing heart, leading to a reduced cardiac field and aberrant heart formation. Reporter gene expression assays provide additional evidence for the repression of Nkx2-5 promoter activity by Shox2. Taken together our results demonstrate that Shox2 plays an essential role in the SAN and pacemaker development by controlling a genetic cascade through the repression of Nkx2-5.

MicroRNAs in Heart Development

MicroRNAs (miRNAs) are a class of small noncoding RNAs of ~22nt in length which are involved in the regulation of gene expression at the posttranscriptional level by degrading their target mRNAs and/or inhibiting their translation. Expressed ubiquitously or in a tissue-specific manner, miRNAs are involved in the regulation of many biological processes such as cell proliferation, differentiation, apoptosis, and the maintenance of normal cellular physiology. Many miRNAs are expressed in embryonic, postnatal, and adult hearts. Aberrant expression or genetic deletion of miRNAs is associated with abnormal cardiac cell differentiation, disruption of heart development, and cardiac dysfunction. This chapter will summarize the history, biogenesis, and processing of miRNAs as well as their function in heart development, remodeling, and disease.

Ectopic expression of Nkx2.5 suppresses the formation of the sinoatrial node in mice.

The sinoatrial node (SAN), functionally known as the pacemaker, regulates the cardiac rhythm or heartbeat. Several genes are expressed in the developing SAN and form a genetic network regulating the fate of the SAN cells. The short stature homeobox gene Shox2 is an important player in the SAN genetic network by regulating the expression of different cardiac conduction molecular markers including the early cardiac differentiation marker Nkx2.5. Here we report that the expression patterns of Shox2 and Nkx2.5 are mutually exclusive from the earliest stages of the venous pole and the SAN formation. We show that tissue specific ectopic expression of Shox2 in the developing mouse heart downregulates the expression of Nkx2.5 and causes cardiac malformations; however, it is not sufficient to induce a SAN cell fate switch in the working myocardium. On the other hand, tissue specific overexpression of Nkx2.5 in the heart leads to severe hypoplasia of the SAN and the venous valves, dis-regulation of the SAN genetic network, and change of the SAN cell fate into working myocardium, and causes embryonic lethality, recapitulating the phenotypes including bradycardia observed in Shox2(-/-) mutants. These results indicate that Nkx2.5 activity is detrimental to the normal formation of the SAN. Taken together, our results demonstrate that Shox2 downregulation of Nkx2.5 is essential for the proper development of the SAN and that Shox2 functions to shield the SAN from becoming working myocardium by acting upstream of Nkx2.5.

Functional Redundancy between Human SHOX and Mouse Shox2 Genes in the Regulation of Sinoatrial Node Formation and Pacemaking Function

The homeodomain transcription factor Shox2 plays a crucial regulatory role in the development of sinoatrial node (SAN) by repressing the expression of Nkx2.5, as demonstrated by failed differentiation of SAN in Shox2 null mice. The SHOX (short stature homeobox) gene family consists of two closely related members, SHOX and SHOX2 in humans, but a SHOX ortholog does not exist in the mouse genome. These two genes exhibit overlapping and distinct expression patterns in many developing organs but whether they share functional redundancy is not known. In this study, we set to investigate possible functional redundancy between SHOX and SHOX2 in vitro and in vivo. We first showed that human SHOX and SHOX2 and mouse Shox2 possess similar transcriptional repressive activities in cell cultures, particularly the repressive effects on the Nkx2.5 promoter activity. We further created an SHOX/Shox2 knock-in mouse line (replacement of Shox2 with SHOX, referred as Shox2KI/KI). Mice carrying the hypomorphic Shox2KI+Neo/KI+Neo allele exhibit bradycardia and arrhythmia and die a few days after birth. However, mice carrying the Shox2KI/KI allele grow to adulthood. Physiological, histological, and molecular analyses demonstrate a fully developed SAN and normal pacemaking function in Shox2KI/KI mice. Our results demonstrate a functional redundancy between human SHOX and mouse Shox2 in the regulation of SAN formation and pacemaking function in addition to several other organs. The SHOX/Shox2 dose appears to be critical for normal pacemaking function.

The Role of Shox2 in SAN Development and Function

Embryonic development is a tightly regulated process, and many families of genes functions to provide a regulatory genetic network to achieve such a program. The homeobox genes are an extensive family that encodes transcription factors with a characteristic 60-amino acid homeodomain. Mutations in these genes or in the encoded proteins might result in structural malformations, physiological defects, and even embryonic death. Mutations in the short-stature homeobox gene (SHOX) is associated with idiopathic short stature in humans, as observed in patients with Turner syndrome and/or Leri-Weill dyschondrosteosis. A closely related human homolog, SHOX2, has not been linked to any syndrome or defect so far. In mice, a SHOX ortholog gene is not present in the genome; however, a true SHOX2 ortholog has been identified. Analyses of Shox2 knockout mouse models have showed crucial functions during embryonic development, including limb skeletogenesis, palatogenesis, temporomandibular joint formation, and cardiovascular development. During embryonic cardiac development, Shox2 is restrictedly expressed in the sinus venosus region, including the sinoatrial node (SAN) and the sinus valves. Shox2 null mutant is embryonically lethal due to cardiovascular defects, including a severely hypoplastic SAN and sinus valves attributed to a significantly decreased level of cell proliferation in addition to an abnormal low heartbeat rate (bradycardia). In addition, it has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the SAN fate and thus plays essential roles in its proper formation and differentiation.

Cerberus functions as a BMP agonist to synergistically induce nodal expression during left–right axis determination in the chick embryo

Left‐sided expression of Nodal in the lateral plate mesoderm (LPM) during early embryogenesis is a crucial step in establishing the left–right (L–R) axis in vertebrates. In the chick, it was suggested that chick Cerberus (cCer), a Cerberus/Dan family member, induces Nodal expression by antagonizing bone morphogenetic protein (BMP) activity in the left LPM. In contrast, it has also been shown that BMPs positively regulate Nodal expression in the left LPM in the chick embryo. Thus, it is still unclear how the bilaterally expressed BMPs induce Nodal expression only in the left LPM. In this study, we demonstrate that BMP signaling is necessary and sufficient for the induction of Nodal expression in the chick LPM where the type I BMP receptor‐IB (BMPR‐IB) likely mediates this induction. Tissue grafting experiments indicate the existence of a Nodal inductive factor in the left LPM rather than the presence of a Nodal inhibitory factor in the right LPM. We demonstrate that cCer functions as a BMP agonist instead of antagonist, being able to enhance BMP signaling in cell culture. This conclusion is further supported by the immunoprecipitation assays that provide convincing biochemical evidence for a direct interaction between cCer and BMP receptor. Because cCer is expressed restrictedly in the left LPM, BMPs and cCer appear to act synergistically to activate Nodal expression in the left LPM in the chick. Developmental Dynamics 237:3613–3623, 2008.

Determination of MiRNA Targets in Skeletal Muscle Cells

MicroRNAs (miRNAs) are a class of small ∼22 nucleotide noncoding RNAs which regulate gene expression at the posttranscriptional level by either destabilizing and consequently degrading their targeted mRNAs or by repressing their translation. Emerging evidence has demonstrated that miRNAs are essential for normal mammalian development, homeostasis, and many other functions. In addition, deleterious changes in miRNA expression were associated with human diseases. Several muscle-specific miRNAs, including miR-1, miR-133, miR-206, and miR-208, have been shown to be important for normal myoblast differentiation, proliferation, and muscle remodeling in response to stress. They have also been implicated in various cardiac and skeletal muscular diseases. miRNA-based gene therapies hold great potential for the treatment of cardiac and skeletal muscle diseases. Herein, we describe methods commonly applied to study the biological role of miRNAs, as well as techniques utilized to manipulate miRNA expression and to investigate their target regulation.

Phosphorylation of Shox2 Is Required for Its Function to Control Sinoatrial Node Formation

Background Inactivation of Shox2, a member of the short‐stature homeobox gene family, leads to defective development of multiple organs and embryonic lethality as a result of cardiovascular defects, including bradycardia and severe hypoplastic sinoatrial node (SAN) and sinus valves, in mice. It has been demonstrated that Shox2 regulates a genetic network through the repression of Nkx2.5 to maintain the fate of the SAN cells. However, the functional mechanism of Shox2 protein as a transcriptional repressor on Nkx2.5 expression remains completely unknown. Methods and Results A specific interaction between the B56δ regulatory subunit of PP2A and Shox2a, the isoform that is expressed in the developing heart, was demonstrated by yeast 2‐hybrid screen and coimmunoprecipitation. Western blotting and immunohistochemical assays further confirmed the presence of phosphorylated Shox2a (p‐Shox2a) in cell culture as well as in the developing mouse and human SAN. Site‐directed mutagenesis and in vitro kinase assays identified Ser92 and Ser110 as true phosphorylation sites and substrates of extracellular signal‐regulated kinase 1 and 2. Despite that Shox2a and its phosphorylation mutants possessed similar transcriptional repressive activities in cell cultures when fused with Gal4 protein, the mutant forms exhibited a compromised repressive effect on the activity of the mouse Nkx2.5 promoter in cell cultures, indicating that phosphorylation is required for Shox2a to repress Nkx2.5 expression specifically. Transgenic expression of Shox2a, but not Shox2a‐S92AS110A, mutant in the developing heart resulted in down‐regulation of Nkx2.5 in wild‐type mice and rescued the SAN defects in the Shox2 mutant background. Last, we demonstrated that elimination of both phosphorylation sites on Shox2a did not alter its nuclear location and dimerization, but depleted its capability to bind to the consensus sequences within the Nkx2.5 promoter region. Conclusions Our studies reveal that phosphorylation is essential for Shox2a to repress Nkx2.5 expression during SAN development and differentiation.

Overexpression of constitutively active BMP-receptor-IB in mouse skin causes an ichthyosis-vulgaris-like disease.

The skin is the outer layer of protection against the environment. The development and formation of the skin is regulated by several genetic cascades including the bone morphogenetic protein (BMP) signaling pathway, which has been suggested to play an important role during embryonic organ development. Several skin defects and diseases are caused by genetic mutations or disorders. Ichthyosis is a common genetic skin disorder characterized by dry scaly skin. Loss-of-function mutations in the filaggrin (FLG) gene have been identified as the cause of the ichthyosis vulgaris (IV) phenotype; however, the direct regulation of filaggrin expression in vivo is unknown. We present evidence that BMP signaling regulates filaggrin expression in the epidermis. Mice expressing a constitutively active form of BMP-receptor-IB in the developing epidermis exhibit a phenotype resembling IV in humans, including dry flaky skin, compact hyperkeratosis, and an attenuated granular layer associated with a significantly downregulated expression of filaggrin. Regulation of filaggrin expression by BMP signaling has been further confirmed by the application of exogenous BMP2 in skin explants and by a transgenic model overexpressing Noggin in the epidermis. Our results demonstrate that aberrant BMP signaling in the epidermis causes overproliferation and hyperkeratinization, leading to an IV-like skin disease.