Repressive Interactions Between Transcription Factors Separate Different Embryonic Ectodermal Domains.

The embryonic ectoderm is composed of four domains: neural plate, neural crest, pre-placodal region (PPR) and epidermis. Their formation is initiated during early gastrulation by dorsal-ventral and anterior-posterior gradients of signaling factors that first divide the embryonic ectoderm into neural and non-neural domains. Next, the neural crest and PPR domains arise, either via differential competence of the neural and non-neural ectoderm (binary competence model) or via interactions between the neural and non-neural ectoderm tissues to produce an intermediate neural border zone (NB) (border state model) that subsequently separates into neural crest and PPR. Many previous gain- and loss-of-function experiments demonstrate that numerous TFs are expressed in initially overlapping zones that gradually resolve into patterns that by late neurula stages are characteristic of each of the four domains. Several of these studies suggested that this is accomplished by a combination of repressive TF interactions and competence to respond to local signals. In this study, we ectopically expressed TFs that at neural plate stages are characteristic of one domain in a different domain to test whether they act cell autonomously as repressors. We found that almost all tested TFs caused reduced expression of the other TFs. At gastrulation these effects were strictly within the lineage-labeled cells, indicating that the effects were cell autonomous, i.e., due to TF interactions within individual cells. Analysis of previously published single cell RNAseq datasets showed that at the end of gastrulation, and continuing to neural tube closure stages, many ectodermal cells express TFs characteristic of more than one neural plate stage domain, indicating that different TFs have the opportunity to interact within the same cell. At neurula stages repression was observed both in the lineage-labeled cells and in adjacent cells not bearing detectable lineage label, suggesting that cell-to-cell signaling has begun to contribute to the separation of the domains. Together, these observations directly demonstrate previous suggestions in the literature that the segregation of embryonic ectodermal domains initially involves cell autonomous, repressive TF interactions within an individual cell followed by the subsequent advent of non-cell autonomous signaling to neighbors.

Six1 proteins with human branchio-oto-renal mutations differentially affect cranial gene expression and otic development.

Single-nucleotide mutations in human SIX1 result in amino acid substitutions in either the protein-protein interaction domain or the homeodomain, and cause ∼4% of branchio-otic (BOS) and branchio-oto-renal (BOR) cases. The phenotypic variation between patients with the same mutation, even within affected members of the same family, make it difficult to functionally distinguish between the different SIX1 mutations. We made four of the BOS/BOR substitutions in the Xenopus Six1 protein (V17E, R110W, W122R, Y129C), which is 100% identical to human in both the protein-protein interaction domain and the homeodomain, and expressed them in embryos to determine whether they cause differential changes in early craniofacial gene expression, otic gene expression or otic morphology. We confirmed that, similar to the human mutants, all four mutant Xenopus Six1 proteins access the nucleus but are transcriptionally deficient. Analysis of craniofacial gene expression showed that each mutant causes specific, often different and highly variable disruptions in the size of the domains of neural border zone, neural crest and pre-placodal ectoderm genes. Each mutant also had differential effects on genes that pattern the otic vesicle. Assessment of the tadpole inner ear demonstrated that while the auditory and vestibular structures formed, the volume of the otic cartilaginous capsule, otoliths, lumen and a subset of the hair cell-containing sensory patches were reduced. This detailed description of the effects of BOS/BOR-associated SIX1 mutations in the embryo indicates that each causes subtle changes in gene expression in the embryonic ectoderm and otocyst, leading to inner ear morphological anomalies.

Six1 and Irx1 have reciprocal interactions during cranial placode and otic vesicle formation.

The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.

A re-examination of lens induction in chicken embryos: in vitro studies of early tissue interactions.

Early studies on lens induction suggested that the optic vesicle, the precursor of the retina, was the primary inducer of the lens; however, more recent experiments with amphibians establish an important role for earlier inductive interactions between anterior neural plate and adjacent presumptive lens ectoderm in lens formation. We report here experiments assessing key inductive interactions in chicken embryos to see if features of amphibian systems are conserved in birds. We first examined the issue of specification of head ectoderm for a lens fate. A large region of head ectoderm, in addition to the presumptive lens ectoderm, is specified for a lens fate before the time of neural tube closure, well before the optic vesicle first contacts the presumptive lens ectoderm. This positive lens response was observed in cultures grown in a wide range of culture media. We also tested whether the optic vesicle can induce lenses in recombinant cultures with ectoderm and find that, at least with the ectodermal tissues we examined, it generally cannot induce a lens response. Finally, we addressed how lens potential is suppressed in non-lens head ectoderm and show an inhibitory role for head mesenchyme. This mesenchyme is infiltrated by neural crest cells in most regions of the head. Taken together, these results suggest that, as in amphibians, the optic vesicle cannot be solely responsible for lens induction in chicken embryos; other tissue interactions must send early signals required for lens specification, while inhibitory interactions from mesenchyme suppress lens-forming ability outside of the lens area.

The pattern of protein and glycoprotein synthesis in presumptive lens and non-lens ectoderm of the chicken embryo.

Lens induction is a classic example of the tissue interactions that lead to cell specialization during early vertebrate development. Previous studies have shown that a large region of head ectoderm, but not trunk ectoderm, of 36 h (stage 10) chicken embryos retains the potential to form lenses and synthesize the protein δ-crystallin under some conditions. We have used polyacrylamide gel electrophoresis and fluorography to examine protein and glycoprotein synthesis in presumptive lens ectoderm and presumptive dorsal (trunk) epidermis to look for differentiation markers for these two regions prior to the appearance of δ-crystallin at 50 h. Although nearly all of the proteins incorporating3H-leucine were shared by presumptive lens ectoderm and trunk ectoderm, these two regions showed more dramatic differences in the incorporation of3H-sugars into glycoproteins. when non-lens head ectoderm that has a capacity for lens formation in vitro was labeled, a hybrid pattern of glycoprotein synthesis was discovered: glycoproteins found in either presumptive lens ectoderm or trunk ectoderm were oftentimes also found in other head ectoderm. Therefore, molecular markers have been identified for three regions of ectoderm committed to different fates (lens and skin), well before features of terminal differentiation begin to appear in the lens.

HATCHING OF ILYANASSA OBSOLETA EMBRYOS: DEGRADATION OF THE EGG CAPSULE PLUG IN THE ABSENCE OF DETECTABLE PROTEOLYSIS OF THE MAJOR PLUG PROTEINS.

The mechanism of action of the hatching substance released by Ilyanassa obsoleta embryos was examined by studying the sequential ultrastructural and biochemical changes that occur in the egg capsule plug as it is dissolved during hatching. When release of the hatching substance is triggered by incubating prehatching embryos in KCl, the inner two layers of the capsule wall (L3 and L4) that extend into the apex to form the plug separate from one another, but only in this region. The hatching substance then dissolves material at the periphery of the plug so that an intact plug can be recovered. However, if the plug is left in contact with the hatching substance, both the thin, electron dense material of the inner layer (L4) and the 10 nm filaments of the adjacent layer (L3) that compose most of the plug are dissolved. The first step in the hatching sequence is mimicked by papain so that L3 and L4 can be separated for analysis on SDS-polyacrylamide gels. L3 contains four major proteins with molecular weights of 24,000-52,000 daltons while L4 contains a predominant 25,000 dalton protein. When isolated plugs are dissolved in crude preparations of the hatching substance and analyzed by polyacrylamide gel electrophoresis, there is evidence of only slight disappearance of one minor plug protein. Based on these findings, Ilyanassa embryos probably release several activities necessary to dissolve the plug, yet degradation of the plug occurs without hydrolysis of the major plug proteins.

Developmental alterations in the appearance of hatching activity inIlyanassa obsoleta embryos.

Embryos of the marine gastropodIlyanassa obsoleta are enclosed within a maternally-produced egg capsule for the first eight days of development. Embryos escape from the capsule by releasing a hatching substance that dissolves the egg capsule plug found at the apex of the capsule. Although hatching was determined to occur at 171 h of development, hatching activity could be detected in homogenates of 96 h embryos and premature hatching of embryos was initiated as early as 90 h of development when capsules were incubated in KCl. Therefore, the hatching substance is stored for over 80 h before its release. Appearance of hatching activity during the fourth day of development required that transcription and then translation occur between 70-76 h of development. Inhibition of either of these processes after 76 h did not prevent the appearance of hatching activity on schedule. In addition, removal of the third polar lobe formed at first cleavage, reduced the quantity of the hatching substance present in day eight lobeless embryos, when compared with control embryos. These results demonstrate a requirement for embryonic gene activity as well as maternal information placed in the egg during oogenesis and segregated into the polar lobe during cleavage for appearance of hatching activity in theIlyanassa embryo.

FORMATION, ORGANIZATION, AND COMPOSITION OF THE EGG CAPSULE OF THE MARINE GASTROPOD, ILYANASSA OBSOLETA.

Embryos of the marine mud snail Ilyanassa obsoleta undergo early development within an egg capsule. After about a week of encapsulation, embryos hatch by releasing a chemical substance that removes the plug found at the apex of a capsule. However, the mechanism of action of this hatching substance remains poorly understood. To study how the hatching substance functions, we examined the composition of the egg capsule, particularly the plug region, to determine what the "substrate" of the hatching substance might be. We have also examined the formation and organization of the egg capsule to determine the origin and identity of the regions of a capsule that the hatching substance must remove. The results show that the Ilyanassa egg capsule is organized into four layers, the outer three of which are composed of protein and carbohydrate. Portions of the two inner layers of the capsule wall extend into the capsule apex and form the plug, which is dissolved by the hatching substance. The isolated capsule plug region contains three major glycoproteins resolved on sodium dodecyl sulfate-polyacrylamide gels. Therefore, the hatching substance may be a protease similar in action to the enzymes released by many other embryos at hatching.