A 3D atlas of the human developing pancreas to explore progenitor proliferation and differentiation.

AIMS/HYPOTHESIS: Rodent pancreas development has been described in great detail. On the other hand, there are still gaps in our understanding of the developmental trajectories of pancreatic cells during human ontogenesis. Here, our aim was to map the spatial and chronological dynamics of human pancreatic cell differentiation and proliferation by using 3D imaging of cleared human embryonic and fetal pancreases. METHODS: We combined tissue clearing with light-sheet fluorescence imaging in human embryonic and fetal pancreases during the first trimester of pregnancy. In addition, we validated an explant culture system enabling in vitro proliferation of pancreatic progenitors to determine the mitogenic effect of candidate molecules. RESULTS: We detected the first insulin-positive cells as early as five post-conceptional weeks, two weeks earlier than previously observed. We observed few insulin-positive clusters at five post-conceptional weeks (mean ± SD 9.25±5.65) with a sharp increase to 11 post-conceptional weeks (4307±152.34). We identified a central niche as the location of onset of the earliest insulin cell production and detected extra-pancreatic loci within the adjacent developing gut. Conversely, proliferating pancreatic progenitors were located in the periphery of the epithelium, suggesting the existence of two separated pancreatic niches for differentiation and proliferation. Additionally, we observed that the proliferation ratio of progenitors ranged between 20% and 30%, while for insulin-positive cells it was 1%. We next unveiled a mitogenic effect of the platelet-derived growth factor AA isoform (PDGFAA) in progenitors acting through the pancreatic mesenchyme by increasing threefold the number of proliferating progenitors. CONCLUSIONS/INTERPRETATION: This work presents a first 3D atlas of the human developing pancreas, charting both endocrine and proliferating cells across early development.

A tridimensional atlas of the developing human head.

The evolution and development of the head have long captivated researchers due to the crucial role of the head as the gateway for sensory stimuli and the intricate structural complexity of the head. Although significant progress has been made in understanding head development in various vertebrate species, our knowledge of early human head ontogeny remains limited. Here, we used advanced whole-mount immunostaining and 3D imaging techniques to generate a comprehensive 3D cellular atlas of human head embryogenesis. We present detailed developmental series of diverse head tissues and cell types, including muscles, vasculature, cartilage, peripheral nerves, and exocrine glands. These datasets, accessible through a dedicated web interface, provide insights into human embryogenesis. We offer perspectives on the branching morphogenesis of human exocrine glands and unknown features of the development of neurovascular and skeletomuscular structures. These insights into human embryology have important implications for understanding craniofacial defects and neurological disorders and advancing diagnostic and therapeutic strategies.

A human embryonic limb cell atlas resolved in space and time.

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months1. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common2. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

Phox2a Defines a Developmental Origin of the Anterolateral System in Mice and Humans.

Anterolateral system neurons relay pain, itch, and temperature information from the spinal cord to pain-related brain regions, but the differentiation of these neurons and their specific contribution to pain perception remain poorly defined. Here, we show that most mouse spinal neurons that embryonically express the autonomic-system-associated Paired-like homeobox 2A (Phox2a) transcription factor innervate nociceptive brain targets, including the parabrachial nucleus and the thalamus. We define the Phox2a anterolateral system neuron birth order, migration, and differentiation and uncover an essential role for Phox2a in the development of relay of nociceptive signals from the spinal cord to the brain. Finally, we also demonstrate that the molecular identity of Phox2a neurons is conserved in the human fetal spinal cord, arguing that the developmental expression of Phox2a is a prominent feature of anterolateral system neurons.