Spatial Transcriptomics for the Analysis of Human Pituitary Development

The pituitary develops from oral ectoderm in contact with the adjacent hypothalamus. However, the precise mechanisms underlying pituitary development in concert with plural tissues are not fully understood, especially in human. A protocol to induce pituitary cells from human induced pluripotent stem cells (hiPSCs) has been established and applied to study pituitary development and disorders. In the method, oral ectoderm and hypothalamus are induced in one organoid, which enables recapitulation of the interactions between these tissues during embryonic development. It leads to self-organization of pituitary cells. Recently, spatial transcriptome technology has been developed and is suitable for the analysis of tissue interactions. Here, we utilized spatial transcriptomics to analyze pituitary organoids, especially focusing on the mechanisms regulating pituitary progenitor cell differentiation. Spatial transcriptomics revealed that the organoids consisted of several cell populations including hypothalamus, oral ectoderm, neural retina, and cortex neuron cells. Pituitary progenitor cells, characterized by the upregulation of LHX3, were included as part of the oral ectoderm population. Further analysis of the population identified human pituitary progenitor-specific genes including many causal genes for congenital hypopituitarism (CPH). Finally, using spatially resolved gene expression data, we examined the hypothalamic population that was in contact with pituitary progenitor cells and identified hypothalamic factors that might regulate progenitor cell differentiation in a paracrine manner. The genes upregulated in the pituitary progenitor and neighboring hypothalamus cell populations are potential causal gene candidates for CPH. In conclusion, spatial transcriptomics provides a novel platform to analyze tissue interaction networks during human pituitary development.

Oral and Palatal Dentition of Axolotl Arises From a Common Tooth-Competent Zone Along the Ecto-Endodermal Boundary

Vertebrate dentitions arise at various places within the oropharyngeal cavity including the jaws, the palate, or the pharynx. These dentitions develop in a highly organized way, where new tooth germs are progressively added adjacent to the initiator center, the first tooth. At the same time, the places where dentitions develop house the contact zones between the outer ectoderm and the inner endoderm, and this colocalization has instigated various suggestions on the roles of germ layers for tooth initiation and development. Here, we study development of the axolotl dentition, which is a complex of five pairs of tooth fields arranged into the typically tetrapod outer and inner dental arcades. By tracking the expression patterns of odontogenic genes, we reason that teeth of both dental arcades originate from common tooth-competent zones, one present on the mouth roof and one on the mouth floor. Progressive compartmentalization of these zones and a simultaneous addition of new tooth germs distinct for each prospective tooth field subsequently control the final shape and composition of the axolotl dentition. Interestingly, by following the fate of the GFP-labeled oral ectoderm, we further show that, in three out of five tooth field pairs, the first tooth develops right at the ecto-endodermal boundary. Our results thus indicate that a single tooth-competent zone gives rise to both dental arcades of a complex tetrapod dentition. Further, we propose that the ecto-endodermal boundary running through this zone should be accounted for as a potential source of instruction factors instigating the onset of the odontogenic program.

SAT-291 SIX3 Is Essential for Hypothalamic and Pituitary Development

The genetic basis for congenital hypopituitarism and related disorders is beginning to emerge, and over causal 30 genes have been identified, including six in the SHH signaling pathway. Mutations in some of these genes can also cause holoprosencephaly (HPE) or septo-optic dysplasia. SIX3 is a homeodomain protein expressed in the developing brain, pituitary gland, and eye. It activates SHH signaling and represses BMP signaling. Heterozygous mutations in SIX3 cause variable HPE in humans and mice. We identified a rare, heterozygous variant in SIX3 in two children with neonatal GH and TSH deficiency and stalk interruption, p.P74R. Using transient transfection in 3T3 cells, we demonstrated that the variant reduced the ability of SIX3 to transactivate the SHH enhancer and promoter of FOXG1, suggesting that the variant could be deleterious. To understand the role of SIX3 in hypothalamic and pituitary development we used Nkx2.1-cre and Prop1-cre to delete Six3 in mice. The Nkx2.1-cre, Six3flox/flox embryos had no evidence of infundibulum evagination or expression of Fgf10 or Tcf7l2 at e11.5. The oral ectoderm invaginated in mutants, but no definitive Rathke’s pouch formed. There was no evidence of Lhx3 expression and only trace amounts of Pitx1, indicating that pituitary induction failed due to the lack of Six3 in the developing hypothalamus. Similarly, disruption of Six3 expression in Rathke’s pouch using Prop1-cre ablated pituitary development. Together, these data reveal essential roles of Six3 in both the neural and oral ectoderm for hypothalamic and pituitary development, respectively. Heterozygous loss of function variants in SIX3 could be a contributor to multiple pituitary hormone deficiencies in children, especially if there are associated craniofacial abnormalities.

Molecular patterning during the development of Phoronopsis harmeri reveals similarities to rhynchonelliform brachiopods

Background Phoronids, rhynchonelliform and linguliform brachiopods show striking similarities in their embryonic fate maps, in particular in their axis specification and regionalization. However, although brachiopod development has been studied in detail and demonstrated embryonic patterning as a causal factor of the gastrulation mode (protostomy vs deuterostomy), molecular descriptions are still missing in phoronids. To understand whether phoronids display underlying embryonic molecular mechanisms similar to those of brachiopods, here we report the expression patterns of anterior (otx, gsc, six3/6, nk2.1), posterior (cdx, bra) and endomesodermal (foxA, gata4/5/6, twist) markers during the development of the protostomic phoronid Phoronopsis harmeri. Results The transcription factors foxA, gata4/5/6 and cdx show conserved expression in patterning the development and regionalization of the phoronid embryonic gut, with foxA expressed in the presumptive foregut, gata4/5/6 demarcating the midgut and cdx confined to the hindgut. Furthermore, six3/6, usually a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria. Surprisingly, brachyury, a transcription factor often associated with gastrulation movements and mouth and hindgut development, seems not to be involved with these patterning events in phoronids. Conclusions Our description and comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarity to that of rhynchonelliform brachiopods, which is likely related to their shared protostomic mode of development. Despite these similarities, the phoronid Ph. harmeri also shows particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

Molecular patterning during the development of Phoronopsis harmeri reveals similarities to rhynchonelliform brachiopods

BackgroundAnswering the question how conserved patterning systems are across evolutionary lineages requires a broad taxon sampling. Phoronid development has previously been studied using fate mapping and morphogenesis, yet molecular descriptions are missing. Here we report the expression patterns of the evolutionarily conserved anterior (otx, gsc, six3/6, nk2.1), posterior (cdx, bra) and endomesodermal (foxA, gata4/5/6, twist) markers in the phoronid Phoronopsis harmeri.ResultsThe transcription factors foxA, gata4/5/6 and cdx show conserved expression in patterning the development and regionalization of the phoronid embryonic gut, with foxA expressed in the presumptive foregut, gata4/5/6 demarcating the midgut and cdx confined to the hindgut. Surprisingly, brachyury, an evolutionary conserved transcription factor often associated with gastrulation movements and patterning of the mouth and hindgut, seems to be unrelated with gastrulation and mouth patterning in phoronids. Furthermore, six3/6, a well-conserved anterior marker, shows a remarkably dynamic expression, demarcating not only the apical organ and the oral ectoderm, but also clusters of cells of the developing midgut and the anterior mesoderm, similar to what has been reported for brachiopods, bryozoans and some deuterostome Bilateria.ConclusionsOur comparison of gene expression patterns with other studied Bilateria reveals that the timing of axis determination and cell fate distribution of the phoronid shows highest similarities to rhynchonelliform brachiopods. Despite these similarities, the phoronid P. harmeri shows also particularities in its development, which hint to divergences in the arrangement of gene regulatory networks responsible for germ layer formation and axis specification.

Panhypopituitarism

Hypopituitarism is defined as a deficiency in 1 or more pituitary hormones. The pituitary gland is composed of the anterior pituitary, which originates from an invagination of the oral ectoderm and forms the Rathke pouch, and the posterior pituitary, which is derived from the neural ectoderm of the diencephalon. The anterior pituitary is composed of 5 types of hormone-producing cells: Somatotrophs produce growth hormone; gonadotrophs, follicle-stimulating hormone and luteinizing hormone; thyrotrophs, thyrotropin; 4 lactotrophs, prolactin; and corticotrophs, corticotropin. Identification of hypopituitarism is important because of its association with premature death due to respiratory and cardiovascular complications.

Acquisition of the dorsal structures in chordate amphioxus

Acquisition of dorsal structures, such as notochord and hollow nerve cord, is likely to have had a profound influence upon vertebrate evolution. Dorsal formation in chordate development thus has been intensively studied in vertebrates and ascidians. However, the present understanding does not explain how chordates acquired dorsal structures. Here we show that amphioxus retains a key clue to answer this question. In amphioxus embryos, maternal nodal mRNA distributes asymmetrically in accordance with the remodelling of the cortical cytoskeleton in the fertilized egg, and subsequently lefty is first expressed in a patch of blastomeres across the equator where wnt8 is expressed circularly and which will become the margin of the blastopore. The lefty domain co-expresses zygotic nodal by the initial gastrula stage on the one side of the blastopore margin and induces the expression of goosecoid , not-like, chordin and brachyury1 genes in this region, as in the oral ectoderm of sea urchin embryos, which provides a basis for the formation of the dorsal structures. The striking similarity in the gene regulations and their respective expression domains when comparing dorsal formation in amphioxus and the determination of the oral ectoderm in sea urchin embryos suggests that chordates derived from an ambulacrarian-type blastula with dorsoventral inversion.