NEUROD2 function is dispensable for human pancreatic β cell specification

Introduction The molecular programs regulating human pancreatic endocrine cell induction and fate allocation are not well deciphered. Here, we investigated the spatiotemporal expression pattern and the function of the neurogenic differentiation factor 2 (NEUROD2) during human endocrinogenesis. Methods Using Crispr-Cas9 gene editing, we generated a reporter knock-in transcription factor (TF) knock-out human inducible pluripotent stem cell (iPSC) line in which the open reading frame of both NEUROD2 alleles are replaced by a nuclear histone 2B-Venus reporter (NEUROD2nVenus/nVenus). Results We identified a transient expression of NEUROD2 mRNA and its nuclear Venus reporter activity at the stage of human endocrine progenitor formation in an iPSC differentiation model. This expression profile is similar to what was previously reported in mice, uncovering an evolutionarily conserved gene expression pattern of NEUROD2 during endocrinogenesis. In vitro differentiation of the generated homozygous NEUROD2nVenus/nVenus iPSC line towards human endocrine lineages uncovered no significant impact upon the loss of NEUROD2 on endocrine cell induction. Moreover, analysis of endocrine cell specification revealed no striking changes in the generation of insulin-producing b cells and glucagon-secreting a cells upon lack of NEUROD2. Discussion Overall, our results suggest that NEUROD2 is expendable for human b cell formation in vitro.


Introduction
Diabetes mellitus results from the loss or progressive dysfunction of insulin-producing pancreatic b cells that leads to hyperglycemia and severe micro-and macrovascular secondary complications.One potential strategy to treat diabetes is restoring functional b cell mass by triggering endogenous regeneration or replacing the lost b cells by islets from cadaveric donors or differentiated from human pluripotent stem cells (hPSCs) in vitro (1)(2)(3)(4)(5).The establishment of hPSC differentiation protocols into islet cells requires a thorough understanding of the mechanisms regulating endocrine lineage formation during human development (6)(7)(8).In mouse and human, endocrinogenesis is governed by differentiation of pancreatic progenitors into endocrine progenitors, which give rise to different hormoneproducing endocrine cells, including a cells (glucagon + ), b cells (insulin + ), d-cells (somatostatin + ), PP cells (pancreatic polypeptide + ) and ϵ cells (ghrelin + ) (Figure 1A).This multistep lineage segregation is coordinated by variety of signaling pathways and tightly regulated by gene regulatory networks (9-11).However, it is still not well known which genetic programs orchestrate distinct endocrine cell fate decisions in humans.Thus, the functional impact of different transcription factors (TFs) on human endocrine cell induction and specification requires further investigation.
Neurogenin-3 (NEUROG3 or NGN3) is the master regulator of endocrinogenesis (12) and deletion of this TF results in endocrine cell agenesis (13).Yet, how this TF precisely regulates endocrine cell induction and specification is unclear.One possible scenario is that NGN3 controls the expression of several key endocrine lineage-specific TFs (14, 15).Among them are Neurod1 and Neurod2 that belong to the bHLH Neurod subfamily, which play important roles in tissue differentiation and lineage commitment.NEUROD1 and NEUROD2 share a high level of similarity in their bHLH domain, suggesting potential conserved functions in DNA binding and target gene activation (16-18).NEUROD2 function is important for neuronal fate specification, migration, and axonal navigation during embryonic development (19).Mice lacking Neurod2 exhibit growth arrest, ataxia, and massive granule cell loss in the cerebellar cortex due to reduced expression of neuronal survival factors that eventually lead to early postnatal death of the knock-out animals (20).Additionally, gene polymorphisms of Neurod2 are directly associated with neurocognitive dysfunctions, such as schizophrenia and epilepsy (21).
Despite the well-studied function of Neurod2 in neurogenesis and brain development, the function of this protein in regulating tissue differentiation in other organs, including the pancreas, has remained rudimentary.During mouse pancreas development, Neurod2 is transiently expressed from E12.5 to E17.5, which corresponds to the peak of endocrinogenesis, while Neurod2 is not detectable in the adult pancreas (22).Furthermore, single cell RNA-sequencing (scRNA-seq) has identified the transient expression of Neurod2 in a subset of endocrine precursors during mouse endocrinogenesis (23).However, mice lacking Neurod2 have shown a normal islet cell composition and morphology ( 22).Yet, the spatiotemporal expression pattern of NEUROD2 during human endocrine lineage formation and the function of this gene in regulating human endocrinogenesis has remained unexplored.Here, we investigated the expression and function of NEUROD2 during human endocrinogenesis by leveraging an iPSC differentiation model in vitro.We identified the transient expression of NEUROD2 transcripts in human endocrine progenitors.By generating a NEUROD2 nVenus/nVenus reporter iPSC line, we established a transient endocrine lineage reporter and showed that NEUROD2 is not required for human endocrine cell induction and b cell specification.

Cell sources
Episomal reprogrammed HMGUi001 (24) and HMGUi001-A-8 (25) iPSC line were used.All cell lines have been confirmed to be  Generation of the NEUROD2 nVenus/nVenus iPSC reporter cell line The NEUROD2 locus was targeted by homologous recombination and CRISPR/Cas9 technology using histone 2B (H2B)-Venus-3xHA-tag targeting vector.We cloned the sequence of the (H2B)-Venus-3xHA-tag into a targeting vector and used the 771 bp upstream of the NEUROD2 start codon and 967 bp downstream of the NEUROD2 stop codon as 5′ and 3′ homology arms (HA).A pair of gRNAs introducing dsDNA breaks 3 bp upstream of the start codon and downstream of the stop codon of the NEUROD2 were cloned into the pu6-sgRNA-CAG-Cas9-Venus-bpA expression vector that allowed FACS sorting (26).This vector harboring gRNAs and Cas9 as well as the targeting vector was transfected into HMGUi001-A-8 iPSCs using the standard Lipofectamine transfection protocols.Transfected cells expressing Cas9-Venus were isolated by FACS and different colonies were picked, expanded, and tested by PCR to choose the desired clones.
In vitro differentiation of stem cell-derived islets (SC-islets) iPSCs were cultured on 1:30 diluted Geltrex (Invitrogen, catalog no.A1413302) in StemMACS iPS-Brew medium (Miltenyi Biotec, catalog no.130-104-368).At ~70% confluency, cultures were rinsed with 1× DPBS without Mg 2+ and Ca 2+ (Invitrogen, catalog no.14190) followed by incubation with accutasse (Gibco) for 3 min at 37°C.Single cells were rinsed with iPS-Brew, and spun down at 1200 rpm for 3 min.The resulting cell pellet was suspended in iPS-Brew medium supplemented with Y-27632 (10 mM; Sigma-Aldrich, catalog no.Y0503) and the single-cell suspension was seeded at ~1.5-2×10 5 cells per cm 2 on Geltrex-coated surfaces for maintenance.Cultures used for 3D differentiation were seeded at 4-5 x 10 6 cells per well in ultra-low attachment (ULA) plates, placed in a shaking platform at 60 rpm or 1 x 10 6 cells per ml in a stirring spinner flask (ABLE corporation) stirring at 60 rpm.Cultures were fed every day with iPS-Brew medium.3D differentiations with cells in ULA plates were started 24 h following seeding.For seeded cells in spinner flask, differentiation was started as soon as ~80% or more aggregates achieved a size of ~150 mM to ~200 mM usually 72 h after seeding.

RNA isolation, cDNA preparation and qPCR analysis
Total RNA was extracted from cells with the miRNeasy mini kit (Qiagen).Isolated RNA was reverse transcribed using the SuperScript Vilo cDNA and cDNA synthesis kit (Life Technologies-Thermofisher Scientific).qPCR was performed using predesigned TaqMan ™ probes (Life Technologies) and 20 ng of cDNA per reaction.Each reaction consisted of 4.5 µL cDNA in nuclease-free water, 5 µL TaqMan ™ Advanced master mix (Life Technologies) and 0.5 µL TaqMan probe ™ (Life Technologies).qPCR was performed using QuantStudio 7 Flex (Thermo Fisher Scientific).Ct-values were normalized among samples, transformed to linear expression values, normalized on reference genes and on control samples.Samples were normalized to the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH).The list of used Taqman probes (Applied Biosystems) are provided in Tables S1.

Flow cytometry
Cells undergoing endocrine differentiation were dissociated to a single cell suspension with accutase for 10-20 min at 37°C.The accutase was inactivated and discarded by washing with iPSC Brew media to later spin down at 1200 rpm for 3 min.Cells pellet was washed once with PBS and fixed with 4% paraformaldehyde for 10 min.Cells were then permeabilized with donkey blocking solution (0.1% tween-20, 10% FBS, 0.1% BSA, 0.2% Triton-X100 and 3% donkey serum).Utilizing the same permeabilization solution, cells were stained with primary antibodies (Table S1) for 1 hour at room temperature or at 4°C overnight.If no conjugated antibody was utilized, the protocol continued with incubation of appropriate secondary antibodies for 30 min-1 h at room temperature (Table S1).Cells were washed with PBS three times after antibody incubation.Flow cytometry was performed using FACS-Aria III (BD Bioscience).FACS gating was determined utilizing isotype, secondary only antibody and stained hiPSCs.FACS data were analyzed using FlowJo.

Confocal microscopy and imaging
Cryosections rehydration started by washing 3 times with 1X PBS.For attached cell monolayer, dispersed cells at different stages were first prepared as single cell solution and plated utilizing 8 well m-Slide (ibidi), left overnight allowing attachment to the surface and form a cell monolayer.The Cryosectioned and/or cell monolayer were next permeabilized with 0.1 M glycine and 0.2% Triton X-100 in PBS for 30 min and later blocked in blocking solution (PBS, 0.1% Tween-20, 1% donkey serum, 5% FCS) for 1 h.The sections were later incubated with the primary antibody (Table S1) diluted in the same blocking solution overnight at 4°C.Afterwards, 3 times washing with 1X PBS is undertaken to later incubate with secondary antibodies (Table S1) diluted in blocking solution.Finally, after being incubated during 2-3 h with the 2 nd antibody, sections were stained for DAPI (1:500 in 1X PBS) for 30 min, rinsed and washed 3x with 1X PBS and mounted using our self-made Elvanol.Images were obtained with a Leica microscope of the type DMI 6000 using LAS AF software and were analyzed and quantified using LAS AF and ImageJ software programs.

ScRNA-seq data analysis
Throughout the analysis of scRNA-seq data, Python 3.9.16 was used, in conjunction with Scanpy (version 1.9.3) (https:// github.com/theislab/scanpy)(28).Quality control and normalized scRNA-seq data were obtained from GEO (reference: GSE132188).This data captures four developmental stages of pancreatic tissue (from E12.5 to E15.5) in mouse embryonic pancreatic cells (23).Two cell types, 'Fev+' and 'Ngn3 high EP', were selected and sequestered into a subset, a Principal Component Analysis (PCA) was executed on the subset.Neighbors in the Neurod2 subset were computed using the 'sc.pp.neighbor' function from Scanpy, UMAP representations for the Neurod2 subset were calculated using the sc.tl.umap function of Scanpy.The Neurod2 high subset was selected based on Neurod2 expression of the 'leiden' attribute.Cells were categorized with a 'Neurod2' gene expression of less than or equal to 1 as 'low', and the rest as 'high'.
For assessing differential gene expression, we utilized the tl.rank_genes_groups function's 'wilcoxon' method.Differential genes were filtered using the 'sc.tl.filter_rank_genes_groups' function.Considering the importance of pseudo-batch data and pseudo-duplication in single-cell RNA-seq differential expression testing (29).So, we used the R package Delegate (https://github.com/cancerbits/DElegate) to strictly screen for differential genes.
Overlaps between the Differential upregulated and downregulated genes with the top 1045 of Neurod2 ChIP-Seq target genes set (30) were computed.A Venn diagram illustrating the overlaps between these three gene sets was plotted using the venn3 function.

Statistical analysis
Comparison of three or more datasets was performed using ordinary one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test.All statistics were performed using GraphPad Prism software 9.

Results
To identify the expression pattern of NEUROD2 during human endocrine lineage formation we used a 3D spinner flask in vitro system (Figure 1B) (27).qPCR analysis identified no expression of NEUROD2 mRNA at pluripotency stage in iPSCs.We found a NEUROD2 transcript expression peak at pancreatic progenitor stage 4 (S4) that was reduced at the beginning of endocrine induction at S5.1.However, a second peak of NEUROD2 expression was evident at S5.4, corresponding to the peak of endocrine progenitor formation.The levels of NEUROD2 were further reduced at S5.7 and S6, the stages corresponding to the generation of stem cell-derived hormone + islet cells (SC-islets).In support of the transient expression during endocrine induction, we found no expression of NEUROD2 mRNA in primary adult human islets and the EndoC-bH1 human b cell line (Figure 1C).In comparison, NEUROD1 was expressed at the endocrine progenitor stage and the expression was even higher in S6 SCislets, human primary islets and the EndoC-bH1 cells (Figure 1D).Together, these data demonstrate the transient expression of NEUROD2 in a subset of human endocrine progenitors, similar to the expression pattern that was previously identified during mouse endocrinogenesis (23,31).
The NEUROD2 gene is located in the chromosome 17q12, consisting of 2 exons from which exon 1 is described as a retained intron, and exon 2 is the protein coding region.To uncover the functional impact of NEUROD2 on human endocrinogenesis, we deleted the entire open reading frame of NEUROD2 and replaced it by a histone 2B (H2B)-Venus-3xHAtag sequence in iPSCs using Crispr/Cas9 technology (Figure 2A).To generate the fluorescently reporter iPSC cell line lacking NEUROD2, we used the heterozygous C-peptide-mCherry reporter hiPSC line (HMGUi001-A-8), which allowed us to monitor insulin production and stem cell-derived b cell (SC-b) formation throughout in vitro differentiation (25) (Figure S1A).Genomic PCR analysis confirmed the homologous recombination at the NEUROD2 locus resulting in generation of two homozygous NEUROD2 knock-out H2B-Venus reporter gene knock-in iPSC clones (C89 and C37) (Figure S1B).We further confirmed the deletion of NEUROD2 sequence and in-frame insertion of the H2B-Venus-3xHA-tag reporter using Sanger sequencing (Figure S1C).The obtained homozygous NEUROD2-KO, nuclear H2B-Venus reporter (NEUROD2 nVenus/nVenus ) iPSC clones were negative for mycoplasma and exhibited a normal karyotype (46, XX) (Figure S1D).Bright-field live imaging of the NEUROD2 nVenus/nVenus clones at the pluripotent stage revealed no phenotypic signs of differentiation and expression of H2B-Venus (Figure S1E).This was supported by immunostaining analysis indicating the expression of the pluripotency markers, SOX2 and OCT3/4 (Figure S1F).Furthermore, we tested for the multilineage differentiation potential of the NEUROD2 nVenus/nVenus iPSC clones confirming the generation of endodermal, mesodermal and ectodermal cells (Figure 2B).To assess the effects of NEUROD2 TF knock-out on pancreatic endocrine lineage differentiation, we employed a six-stage (S) 3D spinner-flask differentiation protocol (Figure 1B) (27).FACS analysis and immunostaining revealed the successful formation of definitive endoderm (DE) at S1, pancreatic progenitor (PP2) at S4 and different pancreatic endocrine cells including SC-b (C-PEP + ), SC-a (GCG + ) and SC-d cells (SST + ) at S6 (Figures 2C, D).Because NEUROD2 mRNA expression peaks at the endocrine progenitor stage, we harvested samples from the end of the endocrine induction stage at S5.4 to monitor the expression levels of NEUROD2.qPCR analysis detected no expression of NEUROD2 mRNA in the NEUROD2 nVenus/nVenus endocrine progenitors compared to the wild-type control cells (Figure 2E).Yet, all the clones expressed comparable levels of NEUROD1 mRNA (Figure 2F), indicating no NEUROD1 compensation in the absence of NEUROD2.Next, we analyzed the H2B-Venus reporter expression, which monitors NEUROD2 transcriptional activity.We found emergence of Venus + cells from the S4 until S5.7, with the peak of expression at S5.4 (Figure 2G).This result supports the expression pattern of NEUROD2 mRNA identified by qPCR analysis.Taken together, we successfully engineered NEUROD2 nVenus/nVenus reporter knock-in knock-out iPSCs lines, which show no deficit in pancreatic endocrine lineage formation.
Next, we analyzed the NEUROD2 reporter activity in endocrine progenitors at S5.4.We found cells with bright nuclear Venus signals but expressing low or no NGN3 or NKX2-2 (Figures 3A, B; white arrowheads).In contrast, the major fraction of cells expressing high levels of NGN3 or NKX2-2 showed low or no Venus reporter activity (Figures 3A, B; blue arrowheads).A subset of cells also co-expressed medium levels of Venus with NGN3 or NKX2-2 (Figures 3A, B; yellow arrowheads).This data suggests that NEUROD2 is expressed in a subset of endocrine progenitors and its expression starts after NGN3, similar to what was reported in mouse in vivo (23).The transient expression of NEUROD2 was further confirmed by detecting Venus reporter activity only in a subset of C-PEP + cells at S5.4.A fraction of Venus + cells with bright signals expressed low levels of C-PEP (white arrowheads) and a fraction of those with high C-PEP expression exhibited low Venus signal (yellow arrowheads).However, most of the C-PEP + cells showed no NEUROD2 reporter activity (blue arrowheads) (Figure 3C).This result indicates that at least a fraction of NEUROD2 + cells eventually evolved into b cell fate.Next, we explored the impact of loss of NEUROD2 function on human endocrine lineage induction.Immunostaining and FACS analysis of S4 differentiated cells revealed a comparable number of PP2 cells co-expressing PDX1 and NKX6-1 between the control and NEUROD2 nVenus/nVenus cells (Figures 3D, E).Yet, we found a difference in the rate of PP2 cell formation between C89 and C37 clones, likely due to clone variation (Figure 3E).In support of this, we identified comparable numbers of NGN3 + progenitors between the three clones, indicating no deleterious effect in NGN3-mediated endocrine induction (Figure 3F).The clone C89 but not the C37 showed a slight increase in the numbers of NKX2-2 + cells compared to the control cells.Yet, the rate of NKX2-2 + cells between the two NEUROD2 nVenus/nVenus cells was comparable (Figure 3G).To support this data, we also performed qPCR analysis at the end of endocrine induction at S5.4, which corresponds to the highest number of Venus + reporter positive cells.This analysis revealed comparable levels of key pancreatic and endocrine regulatory TF mRNA expression for PDX1, NKX6-1, NGN3, NKX2-2, PAX4, ARX and FEV between the three clones (Figure 3H).Yet, an increased tendency but non-significant in the expression levels of the d-cell TF HHEX (32) was found in the NEUROD2 nVenus/nVenus cells compared to the control (Figure 3H).These results indicate that NEUROD2 is not required for human endocrine lineage induction.
To discover the function of NEUROD2 on endocrine lineage differentiation, we analyzed the formation of hormone + cells at S5.7.We used immunofluorescence and stained the SC-islets for the panendocrine marker, CHGA, indicating a comparable number of cells expressing this protein between the three cell lines (Figure 4A).Furthermore, qPCR analysis of S5.7 SC-islets indicated comparable levels of CHGA mRNA between the three clones (Figure 4B).Next, we measured the rate of generation of SC-a and SC-b cells using FACS analysis.We found comparable numbers of C-PEP + , GCG + and C-PEP + -GCG + cells between the control and C89 clones.However, C37 cells exhibited a slightly reduced number of C-PEP + cells and increased number of polyhormonal C-PEP + -GCG + cells but no difference in the number of GCG + cells compared to the control and C89 clones (Figures 4C-E).Immunostaining analysis indicated no apparent difference in the SC-a and SC-b cell composition between the three clones (Figure 4F).To further support these data, we performed qPCR analysis of S5.7 cells for endocrine cell markers.We found comparable mRNA expression levels of INS, GCG, PAX4 and ARX between the three clones (Figures 4G-J).We also extended the qPCR analysis to the S6 SC-islets and found no changes in the expression levels of INS and GCG between the three clones (Figures 4K, L).This analysis demonstrates that NEUROD2 function is not required for SC-a and SC-b cell specification.We next explored the molecular signatures that are likely associated with NEUROD2 function during endocrine lineage formation.Because there are no available human datasets highly enriched for endocrine progenitors expressing NEUROD2, we leverage a scRNA-seq of mouse endocrinogenesis in vivo, which contains a high number of endocrine progenitors (Figure 5A).We detected expression of Neurod2 mainly in endocrine progenitors and in a fraction of Fev + cells (Figure 5B).We then selected all the cells at the time of Neurod2 expression along the pseudotime (circle in Figure 5B) and reclustered them into two populations expressing high levels of Neurod2 (Neurod2 high ) and low or no levels of this TF (Neurod2 low/-) (Figures 5C, D).Differential gene expression analysis revealed 365 upregulated and 532 downregulated genes when comparing the Neurod2 high cluster to the Neurod2 low/-cells (Table S2).We performed pathway enrichment analysis of the upregulated genes using the Metascape annotation and analysis resource (33).Most of the upregulated genes were involved in neuronal cell development and differentiation, cell projection and morphogenesis, hormone secretion, exocytosis and cytoskeleton organization (Figure 5E).Among these, we found upregulation of several genes associated with axonal guidance and growth including Plxna3, Efna3, Fez1, Dpysl5, Emb, Arc, Flrt1, Dab1, Olfm1 and Shroom3.Furthermore, several genes involved in cell migration and cytoskeletal remodeling including Kit, Pgf, Slit1, Sdc3, Map1b, Pak3, Arhgef2, Fermt2, Mapre3, Cyfip2 and Scin were upregulated in Neurod2 high cells (Table S2).This analysis demonstrates the expression of Neurod2 at the stage, in which significant changes in cell dynamics occur, and it likely corresponds to the endocrine cell egression stage during islet cell morphogenesis.
To investigate the possible interlink between Neurod2 function and the highly expressed genes in Neurod2 high cells we used a previously reported ChIP-Seq (chromatin-immunoprecipitation and sequencing) dataset of Neurod2 potential target genes in lineages of cortical projection neurons during neurogenesis (30).Comparing the upregulated genes in Neurod2 high cells with the top 1043 Neurod2 potential targets resulted in identification of 49 overlapped genes (Figure 5F; Table S2).Interestingly, several of these genes including Kit, Slit1, Sdc3, Map1b, Dab1, Olfm1, Dpysl5 and Flrt1 are involved in morphogenetic processes (Figure 5G).Among them, Slit1 belongs to Slit family of secreted extracellular matrix proteins, which have been recently reported to regulate endocrine cell morphogenesis during mouse endocrinogenesis (34,35).Therefore, this analysis predicts a possible function of Neurod2 in regulating programs that coordinate endocrine cell morphogenesis.

Discussion
The gene regulatory networks governing human pancreatic endocrine lineage during embryonic development are still not well explored.Here, we analyzed the spatiotemporal expression pattern of NEUROD2 and the functional impact of its deletion during human endocrinogenesis by combining Crispr/Cas9 technology and iPSC differentiation system in vitro.We successfully generated an iPSC reporter line in which the coding sequence of NEUROD2 was replaced by a H2B-Venus sequence that allowed monitoring the NEUROD2 transcriptional activity, while deleting the NEUROD2 gene.The obtained homozygous clones were pluripotent and successfully differentiated toward all three embryonic lineages.Because NEUROD2 is expressed in many other cell types including neurons (20), the generated line is a valuable tool to study the dynamic expression pattern and functional role of NEUROD2 in the development of other human cell types, such as during neurogenesis and intestinal endocrinogenesis.Furthermore, the expression of H2B-Venus reporter in the NEUROD2 nVenus/ nVenus cells enables having a short lineage tracing tool and allows the specific isolation of NEUROD2-expressing cells for further analysis such a gene profiling and proteomics.Our data revealed no significant impact of loss of NEUROD2 on endocrine cells induction.This finding was not surprising as the expression of NEUROD2 initiates after NGN3 induction (23).Additionally, the levels of NEUROD2 has been reduced in mice lacking Nng3 (14), further suggesting the function of NEUROD2 downstream of Ngn3.Our analysis also did not show striking changes in the rate of formation of SC-a and SC-b cells as the two major types of hormone-producing cells in the pancreas.This finding is aligned well with the previous study in which lack of NEUROD2 did not show significant impact on mouse b cell differentiation (31).However, our previous scRNA-seq analysis predicted a potential relationship between endocrine precursors expressing Neurod2 with a b cell fate during mouse endocrinogenesis (23).Therefore, it is possible that the NEUROD2 function is important to regulate only a subset of b cell-specific programs during development and it is not crucial for their overall differentiation.Additionally, the possible functional impact of NEUROD2 loss on b cell formation might have been masked by the function on NEUROD1.Although we did not find increased NEUROD1 expression upon lack of NEUROD2, the high expression levels of NEUROD1 might be sufficient to overcome the possible phenotype resulting from NEUROD2 deletion.In the future, deep gene-regulatory network studies including single-cell transcriptomics and epigenomics (36,37) combined with NEUROD2 target gene profiling in human endocrine lineage, as has been reported for NGN3 (15), may reveal more subtle phenotypes and functions of NEUROD2 in flow sorted cells during human b cell development.Moreover, due to the increased expression levels of genes associated with hormone secretion and exocytosis in Neurod2 high cells, the possible impacts of NEUROD2 on b cell maturation and function still needs to be investigated.
Previous studies have shown the transient and restricted expression of Neurod2 in a subset of murine endocrine progenitors (23,31).Similarly, our data also indicated an increased expression of NEUROD2 mRNA at the peak of NGN3 + endocrine progenitor formation during human endocrinogenesis.This evolutionarily conserved transient expression pattern suggests that NEUROD2 likely functions only for a short time to regulate molecular programs during endocrinogenesis.Importantly, this time window corresponds to the major morphogenetic events initiated by Ngn3 activity resulting in endocrine cell egression from the pancreatic epithelium to form proto-islets (38, 39).In line with this notion, analysis of mouse scRNA-seq data revealed a positive correlation between Neurod2 expression and expression of several genes involved in endocrine cell dynamics such as those regulating cell polarity, migration and cytoskeletal remodeling.Interestingly, our analysis also predicted possible regulatory function of Neurod2 in the expression of some of these genes.This finding suggests that NEUROD2 might be partially involved in orchestrating endocrine cell morphogenetic programs.Future studies using 3D organoids combined with single cell live imaging (40)(41)(42) should address whether NEUROD2 impacts human endocrine cell egression and islet cell morphogenesis.Interestingly, we also found upregulation of genes associated with axonal guidance and growth in Neurod2 high cells.Considering NEUROD2's role in coordinating synaptic innervation, its impact on neuronal excitability, and synaptic function (43,44), it prompts the question of whether this protein plays a role in the establishment of pancreatic islet innervation.In summary, our data provides the expression pattern analysis of NEUROD2 during human pancreas development and uncovers the dispensable function of this TF in regulating SC-a and SC-b cell differentiation in humans.The current differentiation protocols are designed to generate mainly SC-b cells.Therefore, future analysis of the generated clones using protocols to optimally generate other endocrine cells including SC-d and SC-ϵ cells might precisely reveal whether NEUROD2 function regulates the proper segregation of other endocrine cell types in humans.

1 NEUROD2
FIGURE 1 NEUROD2 mRNA is transiently expressed during human endocrinogenesis.(A) Schematic picture of stepwise pancreatic endocrine lineage formation.(B) Schematic representation of human in-vitro endocrine differentiation protocol.Schemes were created with BioRender.com.(C, D) qPCR analysis of NEUROD2 and NEUROD1 expression at different stages of in-vitro endocrine differentiation as well as in adult primary human islets and EndoC-bH1 human b cell line.

4 NEUROD2
FIGURE 4 NEUROD2 function is dispensable for SC-a and SC-b cell differentiation.(A) Representative confocal pictures showing the C-PEP + and CHGA + cells at S5.7.Scale bar, 50 µm.(B) qPCR analysis of CHGA at S5.7.(C) FACS quantification of C-PEP + cells relative to total cells (upper graph) and total endocrine cells (lower graph) at S5.7.(D) FACS quantification of GCG + cells relative to total cells (upper graph) and total endocrine cells (lower graph) at S5.7.(E) FACS quantification of C-PEP + -GCG + cells relative to total cells (upper graph) and total endocrine cells (lower graph) at S5.7.(F) Representative confocal pictures displaying the C-PEP + and GCG + cells at S5.7.Scale bar, 50 µm.(G-J) qPCR analysis of INS, GCG, PAX4 and ARX at S5.7.(K, L) qPCR analysis of INS and GCG at S6.All statistics have been done using one-way ANOVA.Data are represented as mean ± SD.

5
FIGURE 5 Profiling of Neurod2-expressing cells during endocrinogenesis in vivo.(A) UMAP plot of single cells from embryonic (E12.5-E15.5)pancreatic cells from Bastidas-Ponce et al., 2019.(B) UMAP plot displaying the distribution of cells considered as Neurod2 + cells.(C) UMAP plot showing the different expression levels of Neurod2.(D) UMAP plot indicating two clusters based on Neurod2 expression levels (Neurod2 high and Neurod2 low/-).(E) Pathway enrichment analysis of the 365 upregulated genes in Neurod2 high cluster compared to Neurod2 low/-cells.Selected terms are presented.The whole list of the pathway enrichment analysis is provided in Table S2.(F) Venn diagram showing the overlapping between the upregulated and downregulated genes in Neurod2 high cluster with the Neurod2 potential target genes from the Bayam et al., 2015.(G) The list of 49 Neurod2 potential target genes upregulated in Neurod2 high cells.