Abstract
During early vertebrate development, hematopoietic stem cells (HSCs) are produced from a hemogenic endothelium located in the dorsal aorta, before they migrate to a transient niche where they expand, the fetal liver and the caudal hematopoietic tissue (CHT), in mammals and zebrafish, respectively. In zebrafish, previous studies have shown that the extracellular matrix (ECM) around the aorta needs to be degraded to allow HSCs to leave the aortic floor and reach blood circulation. However, the role of the ECM components in HSC specification has never been addressed. We show here that hapln1b, a key component of the ECM is specifically expressed in hematopoietic sites in the zebrafish embryo. Gain- and loss-of-function experiments all resulted in the absence of HSCs in the early embryo, showing that hapln1b is required, at the correct level, to specify HSCs in the hemogenic endothelium. Furthermore, we show that the expression of hapln1b is necessary to maintain the integrity of the ECM through its link domain. In addition, by combining functional analyses and computer modelling, we show that kitlgb interacts with the ECM, to specify HSCs. Overall, we have demonstrated that the ECM is an integral component of the microenvironment as it mediates specific cytokine signaling that is required for normal HSC specification.
Introduction
The emergence of blood cells is a highly conserved process that consists of many different waves in distinct anatomical locations. The first emerging cells in zebrafish and mammals consist of primitive myeloid and erythroid cells 1–4. Definitive hematopoiesis is then initiated by the emergence of the transient erythro-myeloid precursors (EMPs), arising in the yolk sac in mice and humans 1,2, and in the posterior blood island in zebrafish embryos 5. Hematopoietic stem cells (HSCs) are then specified in the aorta-gonads-mesonephros (AGM) region where they form small intra-aortic clusters 6–11. HSCs are derived directly from the aortic hemogenic endothelium (HE), which is specified by the correct balance of several extrinsic factors, such as VEGF, Hedgehog, Notch, BMP and TGFβ signaling 12–16. Zebrafish HSC specification occurs between 32-60 hours post fertilization (hpf), from the HE in the dorsal aortal 17,18. Early HE specification in zebrafish is initiated by the expression of gata2b 19 followed by runx1 12. Zebrafish HSC specification is dependent upon inflammatory cytokines produced by neutrophils 20–22. Macrophage- and vascular-mediated extracellular matrix (ECM) degradation is also necessary to allow HSCs to migrate into the vein 23,24. HSCs will then migrate to the caudal hematopoietic tissue (CHT), where they interact with endothelial cells and significantly expand their initial number 25,26.
However, the exact mechanisms controlling HSC emergence from the HE and their expansion in the CHT remain to be fully characterized. We, and others, have previously published that these two processes are highly dependent upon cytokine signalling. In particular, in zebrafish, we showed the important and non-redundant roles of oncostatin M and kit-ligand b(kitlgb) in this process 25,27. Proteoglycans, a major component of the ECM, have been previously shown to interact with several hematopoietic cytokines (GM-CSF, IL-3 and Kitlg) and to maintain a close proximity between stromal cells, HSCs and cytokines in the niche 28–30. Here, we study the role of hapln1b, an ECM-associated protein, in contributing to this process. We focus on hapln1b, as it was previously shown to be expressed in the vasculature and in hematopoietic tissues 31. Furthermore, this gene was shown to be important for correct vascular development of the tail vasculature 31. In mammals there are HAPLN1, 2, 3 and 4 genes 32, whereas in zebrafish there are hapln1a, 1b, 2, 3 and 4 31. Hapln1 codes for a link protein, required to attach several chondroitin sulfate proteoglycans to the hyaluronic acid (HA) backbone (a ubiquitous glycosaminoglycan (GAG)) to make large, negatively charged, ECM structures 33. In mammals, Hapln1 is a secreted ECM protein that stabilizes aggrecan-hyaluronan complexes and is required for correct craniofacial 34 and neocortex development 35. Loss of HAPLN1 expression promotes melanoma metastasis but is also required to maintain immune cell motility 36. HAPLN1 is also required to maintain lymphatic vessel integrity and reduce endothelial cell permeability preventing visceral metastasis 37. Knock-Out mouse studies have also revealed a role in maintaining perineural nets (PNNs), a hyaluronan backbone mesh-like network of proteins that surround neurons and regulate neuronal plasticity 38 as well as neural differentiation and development 39–41. Furthermore, PNNs are responsible for binding chemorepulsive molecules (such as Semaphorin3a) 42 as well as transcription factors, such as Otx2 that is exchanged between different neural cells to enhance cortical plasticity 43.
Here we show that hapln1b is necessary for mediating kitlgb-kitb interactions, probably by regulating the ECM stiffness, to induce runx1 expression in the HE during HSC specification in the zebrafish embryo. Gain- and loss-of-function of hapln1b resulted in defective hematopoiesis. Therefore, we conclude that this gene is required, at the correct dosage, for the specification of HSCs from the HE and their development after their emergence.
Methods
Zebrafish strains and husbandry
AB* (WT), along with transgenic and mutant strains were kept in a 14/10h light/dark cycle at 28°C 44. We used the following transgenic animals: lmo2:eGFPzf71 45, gata1:DsRedsd2 46, kdrl:eGFPs843 47, kdrl:Hsa.HRAS-mCherrys896 48, cmyb:GFPzf169 49, globin:eGFP 50, sox10:mRFPvu234 51, mpx:GFP 52, mpeg1:mcherrygl23 53.
Whole-mount In Situ hybridization (WISH) staining and analysis
WISH was performed on 4%PFA-fixed embryos at the developmental time points indicated. Digoxygenin labeled probes were synthesized using a RNA Labeling kit (SP6/T7; Roche). RNA probes were generated by linearization of TOPO-TA or ZeroBlunt vectors (Invitrogen) containing the PCR-amplified cDNA sequence. WISH was performed as previously described 54. Phenotypes were scored by comparing expression with siblings. All injections were repeated three independent times. Analysis was performed using R or GraphPad Prism software. Embryos were imaged in 100% glycerol using an Olympus MVX10 microscope. Oligonucleotide primers used for the production of ISH probes are listed in Table S2.
Cell sorting and flow cytometry
Zebrafish transgenic embryos (fifteen to twenty per biological replicate) were incubated in 0.5mg/mL Liberase (Roche) solution and shaken for 90min at 33°C, then dissociated, filtered and resuspended in 0.9x PBS and 1% FCS. Dead cells were labeled and excluded by staining with 5nM SYTOX red (Life Technologies) or 300nM DRAQ7 (Biostatus). Cell sorting was performed using an Aria II (BD Biosciences) or a Biorad S3 (BioRad). Data was analyzed using FlowJo and GraphPad Prism.
Quantitative real-time PCR (qPCR) and qPCR analysis
RNA was extracted using Qiagen RNeasy minikit (Qiagen) and reverse transcribed into cDNA using a Superscript III kit (Invitrogen) or qScript (Quanta Biosciences). qPCR was performed using KAPA SYBR FAST Universal qPCR Kit (KAPA BIOSYSTEMS) and run on a CFX connect Real time system (Bio Rad). All primers are listed in Table S1. Analysis was performed using Microsoft Excel or GraphPad Prism.
Synthesis of full-length mRNA and microinjection
PCR primers to amplify cDNA of interest are listed in Table S2. Kitlgb and kitlga mRNA was synthesized and injected as previously described 25,27. mRNA was reverse transcribed using mMessage mMachine kit SP6 (Ambion) from a linearized pCS2+ vector containing PCR products. Following transcription, RNA was purified by phenol-chloroform extraction. Hapln1b mRNA was injected at 200pg, unless otherwise stated.
Imaging
WISH were imaged using an Olympus MVX10 microscope in 100% glycerol. Fluorescent images were taken with an Olympus IX83 microscope and processed using cell sense dimension software. All images were processed using Adobe Photoshop. Time-lapse imaging was obtained using a Nikon inverted A1r spectral confocal microscope and processed using Fiji and analysed using GraphPad Prism.
Morpholinos
All morpholinos oligonucleotides (MOs) were purchased from Gene Tools and listed in Table S3. MO efficiency was tested using PCR from total RNA extracted from a pool of 8-10 embryos at 24hpf. Hapln1b full-length primers were used to test MO efficiency and are listed in Table S2. Hapln1b morpholino was injected at 3ng, unless otherwise stated. All MOs used in this study are splice blocking MOs.
Structural modelling and electrostatic properties of kitlga and kitlgb
Structural models for zebrafish kitlga and kitlgb were constructed based on a consensus approach employing homology-modeling, template-based structure modelling, and ab initio structure prediction as implemented in i-TASSER, Phyre2, and RaptorX 55–57. Vacuum electrostatic potential calculations were performed and displayed with the built-in module in the program PyMOL v. 2.3 (https://pymol.org/2/). Isoelectric point (pI) calculations were performed via the prot-pI server (https://www.protpi.ch).
Glycosaminoglycan staining
Embryos were fixed in 4% formol (Biosystems) and embedded in paraffin (Haslab). 3μm sections were stained with 10mg/ml Alcian blue (Sigma) and counterstained with Mayer’s hemalun (Merck) to mark glycosaminoglycans.
Results
hapln1b is specifically expressed in early embryonic hematopoietic tissues
Previous studies have shown that hapln1b was expressed in the vasculature at 24-28hpf, and that its loss-of-function resulted in abnormal angiogenesis 31. However, its role during embryonic haematopoiesis has never been investigated. In zebrafish, hapln1b is the only hapln family member to display a vascular and hematopoietic expression pattern (data not shown), despite retaining high sequence identity and conserved peptides capable of forming disulphide bonds (Figure S1A, B). Synteny, phylogeny and sequence identity analyses revealed that hapln1a and hapln1b originate from a duplication of the HAPLN1 gene in mammals (Figure S2A, B). Even across these species, peptides capable of forming disulphide bonds within the link domain have been conserved (Figure S2C). We therefore focused our study upon hapln1b and first examined its expression pattern. We first established that hapln1b is initially expressed from around 12hpf and would therefore not be derived from maternal RNA 58 (Figure S3A). hapln1b was expressed between 20hpf and 26hpf along the aorta, in the developing CHT and in the hypochord, as previously described 31. Further analyses at 26hpf revealed that hapln1b was also expressed in the aorta and vein region, ventral to the notochord (Figure 1Ai). Between 30 and 36hpf the expression begins to decrease, becoming more localized to the CHT, before being completely restricted to the marginal fold at 48hpf (Figure 1A). Expression was also scored in the cardiac precursors at 36hpf and in possible cranial cartilaginous structures (Figure 1Aii, Aiii). By 4 and 5dpf expression was restricted to cranial structures and was absent from hematopoietic tissue (Figure 1B).
We then further analyzed the expression of hapln1b in different cell populations. We sorted endothelial cells from dissected kdrl:eGFP embryos at 26hpf and 48hpf (Figure 1C). As we found by wholemount in situ hybridisation (WISH), hapln1b was enriched in endothelial cells extracted from whole embryos at 26hpf (Figure 1D) but no enrichment was noted at 48hpf (Figure 1E). However, hapln1b was highly enriched in early EMPs (lmo2:GFP;gata1:DsRed double-positive cells at 28hpf 5) and nascent HSCs (kdrl:mCherry;cmyb:GFP double-positive cells at 36hpf 17), compared to whole embryos at 28hpf (Figure 1F), which showed a potential link between hapln1b and embryonic definitive haematopoiesis. Amino acid structural analysis (using SMART, http://smart.embl.de/) revealed that hapln1b contains an IG domain and two hyaluronic acid (HA) link domains (Figure 1F). We next investigated how this ECM protein could interfere with developmental hematopoiesis.
hapln1b is required to specify HSCs from the Hemogenic Endothelium
To further investigate the role of this gene in hematopoiesis, we injected a splice blocking morpholino (MO) for hapln1b which efficiently reduced mRNA levels (Figure S3B, C). Inhibiting hapln1b expression did not affect gata2b expression, the earliest known marker of HE specification 19 (Figure 2A). However, runx1 and cmyb expression was robustly decreased in the aortic region (Figure 2A). Accordingly, additional downstream expression of cmyb at 4dpf in the CHT and rag1 at 4.5dpf in the thymus were also decreased (Figure 2A, B, and C). To further validate the specificity of our MO, we then rescued the loss of runx1 in hapln1b morphants by co-injecting with hapln1b mRNA (Figure S3D). hapln1b-morphants displayed normal HSC specification, although in a number of cases the formation of the CHT was perturbed as seen in the “mild” cases, as previously described 31 (Figure 2D). In a small number of embryos, the vasculature was severely affected as represented in the “severe” phenotype (Figure 2D). We observed no change in the emergence of primitive macrophages or red blood cells as marked by mfap4 and gata1 at 24hpf (Figure 2E), respectively. We did however note a decrease in primitive circulating neutrophils, as marked by mpx (Figure 2E), although neutrophils were still present on the yolk sac. This change in distribution may be due to a loss of function of the vasculature that we observed in Figure 2D. Loss of hapln1b thus results into a specific loss of HSCs but does not impact primitive haematopoiesis. We then investigated the effect of hapln1b overexpression on embryonic haematopoiesis.
hapln1b overexpression is sufficient to reduce HSC emergence and downstream survival
We next overexpressed hapln1b by injecting the full-length mRNA at the one-cell stage and analysed the effect on developmental haematopoiesis. hapln1b overexpression did not change vessel development or early HSC specification as marked by kdrl at 24hpf and runx1 at 28hpf, respectively (Figure 3A). However, we noted a decrease in cmyb signal at 36hpf in the AGM region (Figure 3A). hapln1b overexpression also decreased cmyb at 48hpf in the CHT, suggesting that newly formed HSCs did not colonize this tissue. However, in the AGM, the cmyb signal was also absent, as in non-injected controls, indicating that HSCs were not lodged and were not unable to migrate from the aorta (Figure 3B). Accordingly, rag1 staining in the thymus at 4.5dpf and cmyb in the CHT at 4dpf were also decreased (Figure 3C). To further analyse this loss of cmyb signal, we then imaged double-positive kdrl:mCherry;cmyb:eGFP embryos to examine HSC emergence form the HE 17. hapln1b overexpression significantly reduced the number of double positive nascent HSCs from the AGM region at 36 and 48hpf (Figure 3D, D’). As expected, the number of HSCs present in the CHT niche at 3 and 4dpf was also significantly reduced (Figure 3E, E’). To further examine this loss of cmyb at 36hpf from the HE we used time-lapse confocal microscopy to image double-positive kdrl:mCherry; cmyb:eGFP embryos to examine the endothelial-to-hematopoietic transition in more detail in the aorta. We imaged from 34hpf to 42hpf to observe HSC budding from the HE. We observed clusters of cells in the AGM region of control embryos, preparing to bud and enter circulation (Movie S1). However, in hapln1b-overexpressing embryos, almost no cells seemed to initiate budding. Occasionally, some cmyb:GFP+ cells were detected, but none of them ever underwent endothelial-to-hematopoietic transition (Movie S2). We therefore conclude that hapln1b overexpression is sufficient to prevent the correct HSC budding from the HE. We next investigated how this could be occurring by examining how hapln1b could alter the ECM.
hapln1b modulates the density of the ECM around vasculature
The degradation of ECM by primitive macrophages is an important step for HSC emigration out of the aortic region 23. Therefore, we sought to confirm that hapln1b expression levels are capable of modulating the ECM directly. To achieve this, we examined glycosaminoglycans in the AGM and CHT region by staining embryo sections with alcian blue. Non-injected controls at 30hpf showed a high concentration of ECM surrounding the notochord, but little or no ECM around the artery and vein in the AGM (Figure 4A). hapln1b-moprhants showed less ECM staining, resulting in a change of the shape of the notochord, aorta and vein in the AGM, suggesting that the integrity of the ECM was altered (Figure 4A). In contrast, hapln1b mRNA overexpression resulted in increased ECM staining between the artery and vein and around vessels in the AGM (Figure 4A). Concerning the CHT, the artery and the venous plexus were clearly visible in controls morphants (Figure 4B). However, in hapln1b-morphants, the venous plexus was highly disrupted with no clear boundary between the artery and vein (Figure 4B). hapln1b mRNA overexpression resulted in a slightly distorted CHT with increased alcian blue staining and densely packed venous plexus (Figure 4B). All these results correlate with the importance of hapln1b in coordinating angiogenesis, as described previously 31 and could explain part of our results concerning developing haematopoiesis. We next examined the role that hapln1b plays in cytokine signalling to control this process.
hapln1b mediates kitlgb-kitb interactions that are required for proper HSC specification from the HE
We next attempted to decipher the mechanism by which hapln1b can affect haematopoiesis. Our previous studies indicated that HSCs express kitb and that kiltgb and kitb are required for runx1 expression in the HE at 28hpf 27. As we found a similar decrease in runx1 in hapln1b-morphants, we investigated a possible link between kiltgb signalling and hapln1b. Previous studies have indicated that kitlgb is expressed in the CHT region by ISH at 24hpf and is likely to exist more commonly as the membrane bound form, due to a loss of one of the two cleavage sites 59. Whereas kitlga would represent the more soluble form of this ligand as it retains the two cleavage sites 59. We also investigated the expression of kiltgb in many different FACS sorted cells and found no enrichment in ICM cells sorted from globin:GFP cells at 20hpf, primitive macrophages sorted from mpeg1:mcherry embryos at 24hpf, primitive neutrophils sorted from mpx:eGFP embryos at 24hpf and neural crest cells sorted from sox10:mRFP embryos at 26hpf (Figure S4 A-D). We also found no enrichment of kitlgb in endothelial cells sorted from dissected embryos at 19-20hpf (Figure S4E). However, we found a significant enrichment in tail endothelial cells at 26hpf (Figure S4F), corroborating with the previously published ISH expression pattern of kitlgb 59. We therefore concluded that kiltgb would be present in the AGM at low concentrations and would require an additional element to mediate effective interaction with its receptor to initiate runx1 expression. We tested this hypothesis by attempting to rescue the loss of runx1 observed in hapln1b-morphants and the loss of cmyb observed in hapln1b-overexpressing embryos by injecting kitlgb mRNA.
To verify the specificity of our kitlgb injection, we also injected kitlga mRNA, which does not play any role in HSC specification and development 27. Injection of either kitlga or kitlgb alone, did not alter expression of runx1 at 28hpf, as anticipated (Figure 5A, A’). Injection of the hapln1b-MO gave a similar loss of runx1 at 28hpf (Figure 5A, A’). We then co-injected the hapln1b-MO along with kiltgb mRNA, which in 16/34 embryos resulted in a rescue of the runx1 signal (Figure 5A, A’). As expected, kiltga could not rescue the loss of runx1 in hapln1b-morphants (Figure 5A, A’). We then attempted to rescue the decrease of cmyb signal in the hapln1b-overexpressing embryos by injecting kitlgb mRNA. Compared to non-injected controls, hapln1b mRNA reduced cmyb expression at 36hpf as observed earlier (Figure 5B, B’). Whereas kitlgb injection alone, did not alter the cmyb expression at 36hpf (Figure 5B, B’), co-injection of both hapln1b and kitlgb mRNAs increased cmyb expression to control levels (Figure 5B, B’). Altogether, this data suggests that the dose of hapln1b controls the stiffness of the ECM, therefore controlling the accessibility of kitlgb to the HE.
As proteoglycans are negatively charged, we sought to examine the possible electrostatic compatibility of kitlgb with such a biological environment. In the absence of an experimentally determined structure for zebrafish kitlgb and kitlga, we leveraged available structural information at high resolution on human KITL (SCF) 60,61 and the related hematopoietic cytokines Flt3 ligand and colony stimulating factor 1 62–64, to derive reliable models based on homology-based approaches and ab initio structure prediction.
Our analysis reveals that Kitlgb would be expected to display an overall positive electrostatic potential as manifested by a Z-potential value of +1.2 at physiological pH and as illustrated by extensive patches of positively charged amino acids at the protein surface (Figure 5C). Such physicochemical properties are in sharp contrast to zebrafish Kitlga and human KITL, which are pronouncedly acidic with very similar electrostatic properties characterized by strongly negative electrostatic Z-potentials at physiological pH. As proteoglycans are negatively charged, the overall positively charged Kitlgb, but not the negatively charged Kitlga, would mediate favourable interactions with the ECM in hemogenic and hematopoietic tissues, therefore activating the Kitb receptor, consistent with our findings.
The link domain is necessary for hapln1b functions
Finally, we wanted to verify that hapln1b was interacting with HA by its link domain, to maintain ECM integrity. We cloned a truncated version of the hapln1b mRNA lacking exon 4 and 5 (hapln1bΔ4Δ5) that encode the HA link domain (Figure 6A). Compared to non-injected controls, hapln1bΔ4Δ5 mRNA injected did not reduce cmyb levels at 36hpf whereas wild-type hapln1b mRNA did (Figure 6B, B’), as shown earlier (Figure 3A). We therefore conclude that the link domain is necessary for hapln1b to exert its function, which is to assemble the ECM. We therefore propose the following model, where kitlgb is probably distributed along the aorta, by interacting with the ECM. The ECM structure is maintained by proteoglycans forming links with the HA backbone (mediated by the link domain of hapln1b), allowing kiltgb to reach the AGM. A loss of hapln1b will prevent HA links to be formed and will prevent kitlgb distribution to the AGM, reducing runx1 expression (Figure 6C). When hapln1b is overexpressed, HSC budding is reduced as the HA links are increased, resulting in a tight ECM that would also reduce kitlgb distribution to the AGM. Therefore, a lower concentration of kitlgb is present in the AGM would result in fewer HSCs budding and developing (Figure 6C).
Discussion
We have shown that hapln1b is expressed along the embryonic endothelium prior to HSC emergence, before it becomes restricted to non-hematopoietic tissue at later stages (Figure 1). hapln1b is required, in the correct concentration, to specify HSCs from the HE and to maintain HSC budding and release into circulation. We found that loss of hapln1b results in a loss of runx1 and loss of downstream HSC specification markers (Figure 2A-C). As proteoglycans can bind cytokines, it is possible that hapln1b is responsible for maintaining HA linking to proteoglycans to favor cytokine-receptor interaction. Our previous data has shown that kitlgb-kitb signalling is required for maintaining runx1 expression and HSC specification in the HE 27. However, we, and others 59, have demonstrated that kitlgb is highly expressed in the posterior blood island but not the aortic HE region. When we modulate hapln1b expression, we are able to change the structure of the ECM as the loss of hapln1b affects the ECM/GAG deposition around axial vasculature, as well as in the CHT vessels and affects the overall CHT structure (Figure 4). The disruption of the CHT structure following hapln1b knockdown was also noted in a previous study 31. This, coupled with the fact that we are able to rescue hapln1b morphants with kitlgb mRNA injections (Figure 5A), lead us to conclude that hapln1b mediates HA linking with proteoglycans and is responsible for stabilising the ECM scaffold and distributing kitlgb to the HE (Figure 6C). Our data shows that the ECM likely plays a crucial role in maintaining the signalling environment in close proximity to the AGM to mediate HSC specification from the HE. Furthermore, we show an involvement of the ECM in the CHT, further suggesting that the ECM is a key player in maintaining the embryonic niche to help expanding HSCs. Although we have highlighted kiltgb as a key cytokine that interacts with the ECM, there are likely many others that contribute to the extrinsic signalling microenvironment. Fully characterizing and understanding these molecules will be an important step to improve the currently challenging derivation of HSCs from iPSCs.
In contrast to our loss-of-function assays, overexpression of hapln1b resulted in normal HSC specification as indicated by normal runx1 expression (Figure 3A), but a reduction in HSC emergence and later expansion within hematopoietic tissue, as indicated by a reduction of cmyb expression (Figure 3B). As we found that HSC emergence was reduced, but never completely ablated, we reasoned that overexpression of hapln1b is likely to result in a denser ECM, reducing the accessibility of kitlgb to the hemogenic microenvironment. We confirmed this by rescuing HSC expansion in hapln1b-overexpressing embryos by overexpressing kiltgb mRNA (Figure 5B). Recent mouse studies have indicated that Kitlg is required throughout HSC development in the AGM region 65. This was further corroborated by additional studies showing that c-kit is expressed by proliferating HSCs in the AGM 66 and that Kitlg maintains HSCs in mouse AGM cultures 67. It is therefore likely that reducing the concentration of kitlgb in our hapln1b-overexpressing embryos restricts HSC emergence and decreases downstream survival and HSC maturation (Figure 6C). It is also possible that the ECM is too dense to allow HSC to bud and migrate to the CHT, as previous studies have indicated that ECM degradation is essential to HSC to enter blood circulation 23,24.
A similar role for Hapln1 was also demonstrated in the PNNs in the mouse central nervous system, as it is required to maintain the HA mesh, surrounding neurons 38. These PNNs bind molecules to maintain neuronal plasticity and development, further implicating this gene in maintaining the ECM. Recent studies have identified that HSCs migrate to the fetal niche, the CHT, and expand their initial number in response to a number of cytokines 24–26. It is possible that many cytokine gradients and concentrations are maintained in the fetal niche by ECM components, such as hapln1b. Understanding these in more details will allow to fully characterizing HSC expansion providing alternative methods to improve HSC expansion ex vivo.
Author contributions
SNS performed structural analysis of KITL, kitlga and kitlgb. VB and AA performed Alcian blue stainings on embryo sections. CBM and CP performed all other experiments. CBM and JYB designed the research and wrote the manuscript. SNS and AA edited the manuscript.
Conflict of interests
The authors declare no conflict of interest
Supplementary Tables
Supplementary Figures
Acknowledgements
J.Y.B. was endorsed by a Chair in Life Sciences funded by the Gabriella Giorgi-Cavaglieri Foundation and is also funded by the Swiss National Fund (310030_184814) and the “Fondation Privée des HUG”.
Footnotes
Key Points
hapln1b encodes a structural protein that is absolutely necessary to HSC emergence
the extracellular matrix is not only necessary for normal vasculature development, but also for kitb/kitlgb signaling in the major vessels, to promote definitive hematopoiesis