Frits Meijlink

Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription Factor Shox2 in Sinoatrial and Pacemaking Development

Background— Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results— To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2−/− embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions— From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Expression patterns of the paired-related homeobox genes MHox/Prx1 and S8/Prx2 suggest roles in development of the heart and the forebrain

Prx1 and Prx2 (previously called MHox and S8, respectively) are the members of a small subfamily of vertebrate homeobox genes expressed during embryogenesis from gastrulation onwards. We directly compared the expression domains of the Prx genes in detail in mouse and in addition some aspects of these patterns in chicken. In addition to the superficially similar expression patterns of Prx1 and Prx2 in cranial mesenchyme, limb buds, axial mesoderm, and branchial arches and their derivatives, we detect major differences at many sites particularly in heart and brain. Our analysis indicated in several cases a correlation with regions developing into connective tissues. From at least day 8.5, Prx-1 expression was observed in the heart, initially in the endocardial cushions and later in the developing semilunar and atrioventricular valves. Prx2 develops early on a diffuse myocardial expression pattern and is later higher expressed in the ventricular septum and in particular in the ductus arteriosus. Prx2 is never expressed in the brain, whereas Prx1 is expressed, from at least day 9.5 onwards, in a unique distinct domain in the ventral part of the hypothalamus, as well as in a broader region of the telencephalon.

Retinoids regulate the anterior expression boundaries of 5′ Hoxb genes in posterior hindbrain

We describe the regulatory interactions that cause anterior extension of the mouse 5′ Hoxb expression domains from spinal cord levels to their definitive boundaries in the posterior hindbrain between embryonic day E10 and E11.5. This anterior expansion is retinoid dependent since it does not occur in mouse embryos deficient for the retinoic acid‐synthesizing enzyme retinaldehyde dehydrogenase 2. A retinoic acid response element (RARE) was identified downstream of Hoxb5 and shown to be essential for expression of Hoxb5 and Hoxb8 reporter transgenes in the anterior neural tube. The spatio‐temporal activity of this element overlaps with rostral extension of the expression domain of endogenous Hoxb5, Hoxb6 and Hoxb8 into the posterior hindbrain. The RARE and surrounding sequences are found at homologous positions in the human, mouse and zebrafish genome, which supports an evolutionarily conserved regulatory function.

The mouse homeobox gene, S8, is expressed during embryogenesis predominantly in mesenchyme

The murine S8 gene, originally identified by Kongsuwan et al. [EMBO J. 7(1988)2131-2138] encodes a homeodomain which resembles those of the paired family. We studied the expression pattern during mid-gestation embryogenesis of S8 by in situ hybridization. Expression was detected locally in craniofacial mesenchyme, in the limb, the heart and the somites and sclerotomes all along the axis, and was absent from the central and peripheral nervous system, splanchnopleure, and endodermal derivatives. This pattern differs considerably from that of most previously described homeobox containing genes. By genetic analysis, the gene was located on chromosome 2, about 20 cM from the HOX-4 cluster.

Dorsoventral patterning of the mouse coat by Tbx15.

Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (deH), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (at) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the deH mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in deH that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in deH/deH animals. Construction of a targeted allele of Tbx15 confirmed that the deH phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology.

Planar cell polarity defects and defective Vangl2 trafficking in mutants for the COPII gene Sec24b

Among the cellular properties that are essential for the organization of tissues during animal development, the importance of cell polarity in the plane of epithelial sheets has become increasingly clear in the past decades. Planar cell polarity (PCP) signaling in vertebrates has indispensable roles in many aspects of their development, in particular, controlling alignment of various types of epithelial cells. Disrupted PCP has been linked to developmental defects in animals and to human pathology. Neural tube closure defects (NTD) and disorganization of the mechanosensory cells of the organ of Corti are commonly known consequences of disturbed PCP signaling in mammals. We report here a typical PCP phenotype in a mouse mutant for the Sec24b gene, including the severe NTD craniorachischisis, abnormal arrangement of outflow tract vessels and disturbed development of the cochlea. In addition, we observed genetic interaction between Sec24b and the known PCP gene, scribble. Sec24b is a component of the COPII coat protein complex that is part of the endoplasmic reticulum (ER)-derived transport vesicles. Sec24 isoforms are thought to be directly involved in cargo selection, and we present evidence that Sec24b deficiency specifically affects transport of the PCP core protein Vangl2, based on experiments in embryos and in cultured primary cells.

Targeted inactivation of Hoxb8 affects survival of a spinal ganglion and causes aberrant limb reflexes.

Hoxb8 mutant mice were generated by inserting the lacZ coding sequence in frame with the first exon of Hoxb8. These mice express a fusion protein with a functional beta-galactosidase activity instead of Hoxb8. Mutant embryos were analyzed for anatomical changes. The results indicate that Hoxb8 is not an indispensable regulator of A-P patterning in the forelimb, unlike suggested by our Hoxb8 gain of function experiments (Charité J, DeGraaff W, Shen S, Deschamps J. Cell 1994;78:589-601). The null mutant phenotypic traits include degeneration of the second spinal ganglion (C2), an abnormality opposite to the alteration in the gain of function transgenic mice. Subtle changes in the thoracic part of the vertebral column were observed as well. Adult homozygous mutants exhibit an abnormal clasping reflex of the limbs.

Genetics of shoulder girdle formation: roles of Tbx15 and aristaless-like genes

The diverse cellular contributions to the skeletal elements of the vertebrate shoulder and pelvic girdles during embryonic development complicate the study of their patterning. Research in avian embryos has recently clarified part of the embryological basis of shoulder formation. Although dermomyotomal cells provide the progenitors of the scapular blade, local signals appear to have an essential guiding role in this process. These signals differ from those that are known to pattern the more distal appendicular skeleton. We have studied the impact of Tbx15, Gli3, Alx4 and related genes on formation of the skeletal elements of the mouse shoulder and pelvic girdles. We observed severe reduction of the scapula in double and triple mutants of these genes. Analyses of a range of complex genotypes revealed aspects of their genetic relationship, as well as functions that had been previously masked due to functional redundancy. Tbx15 and Gli3 appear to have synergistic functions in formation of the scapular blade. Scapular truncation in triple mutants of Tbx15, Alx4 and Cart1 indicates essential functions for Alx4 and Cart1 in the anterior part of the scapula, as opposed to Gli3 function being linked to the posterior part. Especially in Alx4/Cart1 mutants, the expression of markers such as Pax1, Pax3 and Scleraxis is altered prior to stages when anatomical aberrations are visible in the shoulder region. This suggests a disorganization of the proximal limb bud and adjacent flank mesoderm, and is likely to reflect the disruption of a mechanism providing positional cues to guide progenitor cells to their destination in the pectoral girdle.

Concerted involvement of Cdx/Hox genes and Wnt signaling in morphogenesis of the caudal neural tube and cloacal derivatives from the posterior growth zone

Decrease in Cdx dosage in an allelic series of mouse Cdx mutants leads to progressively more severe posterior vertebral defects. These defects are corrected by posterior gain of function of the Wnt effector Lef1. Precocious expression of Hox paralogous 13 genes also induces vertebral axis truncation by antagonizing Cdx function. We report here that the phenotypic similarity also applies to patterning of the caudal neural tube and uro-rectal tracts in Cdx and Wnt3a mutants, and in embryos precociously expressing Hox13 genes. Cdx2 inactivation after placentation leads to posterior defects, including incomplete uro-rectal septation. Compound mutants carrying one active Cdx2 allele in the Cdx4-null background (Cdx2/4), transgenic embryos precociously expressing Hox13 genes and a novel Wnt3a hypomorph mutant all manifest a comparable phenotype with similar uro-rectal defects. Phenotype and transcriptome analysis in early Cdx mutants, genetic rescue experiments and gene expression studies lead us to propose that Cdx transcription factors act via Wnt signaling during the laying down of uro-rectal mesoderm, and that they are operative in an early phase of these events, at the site of tissue progenitors in the posterior growth zone of the embryo. Cdx and Wnt mutations and premature Hox13 expression also cause similar neural dysmorphology, including ectopic neural structures that sometimes lead to neural tube splitting at caudal axial levels. These findings involve the Cdx genes, canonical Wnt signaling and the temporal control of posterior Hox gene expression in posterior morphogenesis in the different embryonic germ layers. They shed a new light on the etiology of the caudal dysplasia or caudal regression range of human congenital defects.

Expression patterns of group-I aristaless-related genes during craniofacial and limb development.

Aristaless-related proteins are structurally defined by the presence of a paired-type homeodomain and an additional conserved domain, known as aristaless domain or OAR-domain. These proteins can be further categorized in three groups (Int. J. Dev. Biol., 43 (1999) 651). Group-I aristaless-related genes are linked to functions in the development of the craniofacial and appendicular skeleton and are expressed predominantly in the mesenchyme in stages from gastrulation through at least mid-gestation (Mech. Dev., 48 (1994) 245; Mech. Dev., 52 (1995) 51; Development, 124 (1997) 3999; Dev. Biol., 199 (1998) 11; Development, 126 (1999) 495). In view of the highly redundant character of the functions of these genes in patterning craniofacial and limb structures, we found it important to directly compare their expression patterns at critical stages of craniofacial and limb development.